KR20220148859A - Lymphocyte populations and methods of producing them - Google Patents

Abstract

The present invention relates to novel populations of lymphocytes, methods of producing them, and their use in the treatment of diseases.

Description

Lymphocyte populations and methods of producing them

The present disclosure relates to novel populations of lymphocytes and immune cells, methods of generating them, and their use in the treatment of diseases. More specifically, the present disclosure relates to methods of producing new populations of natural killer T cells (NKT cells), T cells, and dendritic cells using high doses of glucocorticoids and glucocorticoid receptor agonists.

We previously found that high concentrations of glucocorticoids could be used to modulate patients to enhance the efficacy of cellular immunotherapy, such as adaptive T cell therapy; International patent application PCT/US2018/025517 published as WO2018/183927. In that application, the authors noted the toxicity associated with chemotherapy and radiation-mediated pretreatment, which is believed to non-selectively destroy the cellularity of the spleen. The authors provided acute doses of glucocorticoids (a subclass of steroids) and other non-toxic lymphopenic agents to benefit cancer patients receiving cellular immunotherapy.

In International Patent Application PCT/US2019/054395, the authors also described the use of high concentrations of glucocorticoids to induce lymphatic depletion of peripheral blood lymphocytes without substantially affecting the cell number of other cells. In that application, the authors noted that high concentrations of glucocorticoids can deplete peripheral blood lymphocytes, including, for example, islet-specific autoreactive T cells responsible for diabetic autoimmunity, but neutrophils, platelets, red blood cells and stem cells (HSCs and MSCs). all) reported to preserve. The authors have offered glucocorticoids as a non-myelodestructive therapy that can perform safe immunological reprogramming with efficacy comparable to chemotherapy.

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Reducing the use of cytotoxic chemotherapy is a top priority for the National Cancer Institute. Carcinomas, often called solid tumors, account for 80-90% of all cancers, but have proven difficult to target with the development of new cancer therapies. Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable success in the treatment of CD-19 expressing B-cell acute lymphocytic leukemia. However, there are many obstacles limiting CAR T cell therapy for solid tumors: inefficient trafficking to the tumor and the immunosuppressive microenvironment of solid tumors limit T cell efficacy. In addition, CAR T therapy has been associated with serious side effects including cytokine release syndrome (CRS), neuroedema, and graft-versus-host disease (GvHD).

Natural killer T cells (NKTs) are a heterogeneous group of T cells that share the characteristics of T cells and natural killer (NK) cells. Unlike conventional T cells, NKTs mature functionally when they emerge from the thymus and are primed for rapid cytokine production. NKT directly kills CD1d-expressing cancer cells and tumor microenvironment macrophages, rapidly produces and releases immune-activating cytokines such as IFNgamma and IL-4, dendritic cells (DC), NK cells, and B and T lymphocytes. It can activate other immune cells. Clinically, invariant NKT (iNKT) has been shown to activate endogenous NKT by injection of 'autologous culture activated iNKT', administration of alpha Gal Cer (NKT activator) loaded dendritic cells or monocytes, or KRN7000, a synthetic analogue of alpha Gal Cer. It can be used against a variety of different cancers by administering NKT activator antibodies or ligands such as

However, none of these methods used to induce iNKT production have proven effective in cancer patients; iNKT levels decreased in cancer patients and clinical trials were disappointing. iNKT levels are similarly low in the elderly (Tarazona et al, 2003, incorporated herein by reference in its entirety). The use of 'autologous activated NKT' in melanoma was effective in 3 out of 9 patients, and the outcome was directly related to the number of tumor-infiltrating NKTs (Wolf et al , 2018 and Nair et al , 2017, all of them). incorporated herein by reference). However, this approach was limited by the small number of NKTs in cancer patients and the plasticity of iNKTs migrating between IFN-gamma type 1 and tumor-promoting IL-4 type 2 .

In cancer treatment, kinase inhibitors (KI) are well tolerated compared to conventional cytotoxic chemotherapy. However, significant toxicity, including fatigue, hypertension, rash, impaired wound healing, bone marrow suppression, and diarrhea, abnormalities of thyroid function, bone metabolism, linear growth, gonad function, fetal development, adrenal function, and glucose metabolism, is associated with kinase inhibitors. are still related Many patients require dose reduction due to the toxicity of KI, which must be taken chronically (Lodisch et al , 2013, incorporated herein by reference in its entirety). In addition, resistance to KI is general and time-dependent with treatment (Bhullar 2018, incorporated herein by reference in its entirety).

Despite efforts to reduce the toxicities associated with cancer treatment, the physical and medical costs of managing these toxicities remain significant. For example, up to 41% of blood cancer patients choose to stop taking a new kinase/proteasome inhibitor or biologic because of the physical and financial toxicity associated with these drugs (Mato 2018, Kadri 2017, Mato 2016 and Barrett 2010) , each incorporated herein by reference in its entirety).

T cells are a type of lymphocytes that play an important role in the immune response. T cells are distinguished from other types of lymphocytes in that they have T cell receptors on the cell surface. The T-cell receptor (TCR) is responsible for recognizing antigen fragments bound to major histocompatibility complex (MHC) molecules and is a heterodimer of two different protein chains. In 95% of T cells in humans, the TCR consists of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR is composed of gamma and delta (γ) /δ) chains (encoded by TRG and TRD, respectively). This rate varies in diseased conditions (eg leukemia).

In contrast to MHC-restricted alpha beta T cells, gamma delta T cells do not require antigen processing and major histocompatibility complex (MHC) presentation of peptide epitopes for activation, although some recognize MHC class Ib molecules. Some gamma delta T cells recognize markers of cellular stress due to infection or tumorigenesis. Gamma delta T cells are believed to play a role in recognizing lipid antigens.

Gamma delta T cells are recognized, infected or transformed by the production of cytokines (IFN-γ, TNF-α, IL-17) and chemokines (RANTES, IP-10, lymphotactin). It exhibits extensive functional plasticity after cell lysis of target cells (Perforin, Granzyme, TRAIL) and interaction with other cells. Gamma delta T cells can recognize and lyse various cancers in an MHC-unlimited manner, have protective functions in infectious diseases, and have been shown to be associated with the progression and prognosis of various infectious diseases (Gogoi et al, 2013; Pauza). et al, 2018; Zheng et al, 2012; Dong et al, 2018; Zhao et al 2018; all incorporated herein by reference in their entirety). Some gamma delta T cells may also act as antigen presenting cells in some circumstances (Himoudi et al, 2012). Therefore, gamma delta T cells are of considerable interest in immunotherapeutic development.

Dendritic cells are bone marrow-derived leukocytes and are the most potent antigen presenting cells of the mammalian immune system. Dendritic cells are often classified into common dendritic cell (cDC) and plasmacytoid dendritic cell (pDC) subsets. Dendritic cells mainly exist in two basic functional states: "immature" and "mature". Turn on the activating (mature) metabolic, cellular and gene transcriptional programs of dendritic cells, allowing DCs to migrate from peripheral tissues to T-dependent regions of secondary lymphoid organs where T lymphocyte-activated antigen presentation can occur (Patente et al, 2018) ; incorporated herein by reference in its entirety).

The main function of dendritic cells is to process antigenic substances and present them to T cells on the cell surface to initiate an adaptive immune response. Dendritic cells can also promote self-tolerance by producing polarizing cytokines that promote pathogen-specific effector T cell differentiation and activation, and secreting tolerogenic cytokines that induce the differentiation of regulatory T cells. In view of their immunomodulatory function, dendritic cells are of considerable interest in the development of immunotherapy for the treatment of conditions including cancer, autoimmune diseases and infections. For example, CD11b positive dendritic cells are associated with influenza A (H1N1) infection, and reduced severity, or protection, of respiratory syncytial virus (Lee et al, 2018; Malloy et al, 2017; all here in its entirety) incorporated by reference).

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Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In most cases, mild symptoms (which may include fever, cough, and shortness of breath) develop, but some progress to viral pneumonia and multiorgan failure. The COVID-19 outbreak was declared a pandemic by the World Health Organization (WHO) in March 2020. As of April 2020, there have been over 1 million confirmed cases worldwide, resulting in over 50,000 deaths. As of April 2020, there is no vaccine or specific antiviral treatment for COVID-19, and disease management is focused on symptomatic treatment and supportive care.

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There is a need for additional treatments for cancer, autoimmune disorders, and infectious (also called microbial) diseases that are safer and are associated with less toxicity and/or greater efficacy than currently available therapies. A simpler, less toxic and less expensive treatment is needed.

The present invention is based on the surprising discovery that high doses of glucocorticoids act to induce lympho-depletion of many types of peripheral blood lymphocytes, but induce the production/activation/mobilization of new populations of natural killer T (NKT) cells. In addition to presenting known NKT cell properties, this novel population of NKT cells can directly engulf cancer cells, thus expanding the potential of high concentrations of glucocorticoids as therapeutics for solid cancers.

We also found that, after high dose administration, glucocorticoid molecules can bind and block intercellular adhesion molecules such as ICAM3. Binding is cooperative and up to 26 molecules bind to the first Ig domain of ICAM3. ICAM3 is expressed at significant levels in cells such as lymphocytes, monocytes and neutrophils, as well as cancer cell types such as melanoma and osteosarcoma.

Accordingly, in a first aspect, the present invention provides a method for producing a population of natural killer T cells (NKT cells), wherein the method may be a glucocorticoid receptor (GR) modulator or an ICAM3 modulator (a glucocorticoid such as dexamethasone) ) to the subject at a dose equivalent to approximately at least 6 mg/kg human equivalent dose (HED) of dexamethasone base, wherein the glucocorticoid induces a population of NKT cells in the subject. The NKT cells of the present invention exhibit novel patterns of marker expression. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells are CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1 , Ly6G, Sca1, and/or TCR gamma/delta; It is characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta. In some embodiments, the NKT cells express CD3, CD4, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, and Sca1. In some embodiments, the NKT cells express CD3, CD4, CD45, CD56, CD62L, NK1.1, Ly6G, and Sca1. In some embodiments, the NKT cells express CD3, CD4, CD45, CD49b, CD62L, NK1.1, Ly6G, and Sca1. In some embodiments, the NKT cells express CD3, CD4, CD45, CD56, CD62L, NK1.1, and Ly6G. In some embodiments, the NKT cells express CD3, CD4, CD45, CD49b, CD62L, NK1.1, and Ly6G. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells are CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1 , Ly6G, and/or TCR gamma/delta expression; It is characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G , and/or expressing TCR gamma/delta; It is characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta. In some embodiments, the NKT cell does not express C-kit, B220, FoxP3, or TCR alpha/beta. In some embodiments, the NKT cells do not express Sca1. The NKT cells may express CD8. The NKT cells may express CD8. The NKT cells may express CD4. The NKT cells may express CD4. said NKT cells may express CD4 and CD8; Ly6G can be expressed.

In some preferred embodiments the NKT cells of the present disclosure are capable of expressing CD3, CD45, and/or CD56. In some such embodiments, the NKT cells of the present disclosure may be CD3+/bright or CD3+/very bright, and/or CD45+/dark, and/or CD56+.

NKT cells can be described as

CD4+/매우 밝음;

CD4+/very bright;

CD8+/어두움;

CD8+/dark;

CD3+/매우 밝음;

CD3+/very bright;

CD45+/어두움;

CD45+/dark;

Sca1+/매우 밝음;

Sca1+/very bright;

CD44+/-;

CD44+/-;

CD69+/-;

CD69+/-;

CD25+/-;

CD25+/-;

TCR 감마 델타+; 및/또는

TCR gamma delta+; and/or

CDd49b+ 또는 CD56+/밝음.

CDd49b+ or CD56+/bright.

NKT cells can be described as having these properties in naive subjects. NKT cells can be described as having these properties in a tumor/cancerous or autoimmune state.

The expression level of a cellular marker can be determined in relation to the average expression level of a reference NKT cell population derived from a common source not contacted with a glucocorticoid receptor (GR) modulator or an ICAM3 modulator. Expression of a marker can be measured, for example, by flow cytometry performed using the equipment, reagents and/or conditions described herein (taken alone or in combination). The glucocorticoid receptor (GR) modulator or ICAM3 modulator may be a glucocorticoid. In some embodiments, the glucocorticoid is selected from the group consisting of dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide, betamethasone, flumethasone, and beclomethasone.

In a preferred embodiment, the glucocorticoid is selected from the group consisting of dexamethasone, betamethasone, and methylprednisone (preferably dexamethasone or betamethasone).

In some embodiments, the glucocorticoid is dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicotinate, dexamethasone-21-acetate, dexamethasone phosphate, dexamethasone phosphate-21- Phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate, dexamethasone palmitate, dexamethasone-21-palmitate, dexamethasone dipropionate, dexamethasone propionate, dexamethasone acetate anhydrous, dexamethasone acetate 21-phenylpropionate, dexamethasone-21-sulfobenzoate, dexamethasone hemo-sulfate, dexamethasone sulfate, dexamethasone beloxyl, dexamethasone acid, dexamethasone acepurate, dexamethasone sodium carboxyimide, dexamethasone cifesylate, dexamethasone 21-phosphate Salt, dexamethasone mesylate, dexamethasone linoleate, dexamethasone glucoside, dexamethasone glucuronide, dexamethasone iodine acetate, dexamethasone oxetanone, carboxymethylthio-dexamethasone, dexamethasone bisethoxime, dexamethasone epoxide, dexamethasone linoleate Dexamethasone methylorthovalerate, dexamethasone spermine, 6-hydroxy dexamethasone, dexamethasone tributyl acetate, dexamethasone aspartic acid, dexamethasone galactopyranose, dexamethasone hydrochloride, hydroxy dexamethasone, carboxydexamethasone, desoxydexamethasone , dexamethasone cyclodextrin, dihydrodexamethasone, oxodexamethasone, propionyloxy dexamethasone, dexamethasone galactodi, dexamethasone isonicotinate, dexamethasone sodium hydrogen phosphate, dexamethasone aldehyde, dexamethasone cromethonate, dexamethasone tridecylate, Dexamethasone methanesulfonate, dexamethasone butyl acetate, dehydrodexamethasone, dexamethasone isothiocyanatoethyl thioether, dexamethasone bromoacetate, dexamethasone hemiglutarate, deoxydexamethasone, dexamethasone chlorambusylate, dexamethasone melpalanate, formyloxy dexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and any combination treatment containing a form of dexamethasone.

In some embodiments, the glucocorticoid is dexamethasone, which is dexamethasone sodium phosphate.

The methods of the present invention may comprise the administration of specific glucocorticoid doses. In some embodiments, the glucocorticoid is administered at a dose approximately equivalent to:

덱사메타손 염기의 6-12 mg/kg 인간 등가 용량 (HED);

6-12 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 적어도 6 mg/kg 인간 등가 용량 (HED);

at least 6 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 적어도 12 mg/kg 인간 등가 용량 (HED);

at least 12 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 적어도 15 mg/kg 인간 등가 용량 (HED);

at least 15 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 적어도 18 mg/kg 인간 등가 용량 (HED);

at least 18 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 적어도 21 mg/kg 인간 등가 용량 (HED);

at least 21 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 적어도 24 mg/kg 인간 등가 용량 (HED);

at least 24 mg/kg human equivalent dose (HED) of dexamethasone base;

덱사메타손 염기의 최대 45 mg/kg 인간 등가 용량 (HED).

Up to 45 mg/kg human equivalent dose (HED) of dexamethasone base.

In some preferred embodiments, the glucocorticoid is administered at a dose equivalent to approximately at least 18 mg/kg human equivalent dose (HED) of dexamethasone base. In some other preferred embodiments, the glucocorticoid is administered at a dose equivalent to approximately at least 15-18 mg/kg human equivalent dose (HED) of dexamethasone base.

The glucocorticoid dose may be defined as the human equivalent dose (HED) of dexamethasone having a mg/kg value in the range of mg/kg values, wherein the range is two of the mg/kg values given in i) to viii) above. Limited. For example, the glucocorticoid dose can be defined as 6-45 mg/kg of dexamethasone HED. In another example, the glucocorticoid dose may be defined as 12-24 mg/kg of dexamethasone HED.

The glucocorticoids can be administered as a single acute dose or as a total dose for about 72 hours. Moreover, the method may comprise administering one or more additional doses of a glucocorticoid. In some embodiments, one or more additional doses are administered: from 24 hours to 120 hours after the prior glucocorticoid administration; 24-48 hours after prior glucocorticoid administration; 72 to 120 hours after prior glucocorticoid administration; every 24, 48, 72, 96, 120, 144 or 168 hours after the first glucocorticoid administration; Once weekly after the first glucocorticoid administration; Once every 2 weeks after the first glucocorticoid administration; Once a month after the first glucocorticoid administration; or twice weekly after the first glucocorticoid administration.

The present invention may include a step for NKT cell activation. Accordingly, in some embodiments, the method may further comprise administering to the subject a NKT cell activator. The NKT cell activator may be selected from the group consisting of alpha GalCer, sulfide, or NKT-activating antibody. The NKT cell activator may be alpha GalCer loaded dendritic cells or monocytes. The NKT cell activator may be administered within 1-48 hours or about 1-48 hours after administration of the glucocorticoid. The NKT cell activator may be administered within 48 hours or about 48 hours after administration of the glucocorticoid.

In some embodiments, the subject is a mammal, eg, a human.

The subject has or may be suspected or suspected of having (or has been diagnosed with) cancer, an autoimmune disease, or an infectious disease (also called a microbial disease). The cancer may be a solid tumor. Alternatively, the cancer may be a lymphoma, preferably a B-cell lymphoma or a T-cell lymphoma. In some preferred embodiments, the cancer may be non-Hodgkin's lymphoma.

The cancer may be selected from the group consisting of: squamous cell carcinoma (eg epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung; peritoneal cancer; hepatocellular carcinoma; stomach or abdominal cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver tumor; breast cancer; colorectal cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary adenocarcinoma; kidney or kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocarcinoma; anal carcinoma; penile carcinoma; and head and neck cancer.

The NKT cells of the present invention can treat cancer through tumor infiltration. The NKT cells of the present invention can treat cancer through the release of immune activating cytokines. The NKT cells of the present invention can treat cancer and can engulf and kill cancer cells. The NKT cells of the present invention can treat cancer by promoting the infiltration of other immune cells into the tumor. The NKT cells of the present invention can treat cancer through CD1d-induced apoptosis. The NKT cells of the present invention can treat cancer through tumor necrosis. The NKT cells of the present invention can treat cancer by recognizing high levels of phosphate antigen produced by tumor cells through expression of a gamma-delta T cell receptor on the NKT cells of the present invention. Accordingly, in some embodiments, the present invention provides a method of inducing or administering the NKT cells of the present invention to induce tumor necrosis. In some embodiments, the present invention provides a method of inducing CD1d-induced apoptosis of cancer cells by inducing or administering the NKT cells of the present invention. In some embodiments, the invention provides a method of swallowing and/or killing cancer cells using the NKT cells of the invention. In some embodiments, the present invention provides a method of activating gamma-delta expressing NKT cells by a cancer cell phosphate antigen that recognizes and kills cancer cells via NK receptor(s) on NKT cells.

In embodiments wherein the subject has, is suspected of having (or has been diagnosed with) an autoimmune disease, the autoimmune disease is multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes mellitus (T1D), scleroderma , pemphigus, or lupus. In embodiments where the subject has or is suspected of having (or has been diagnosed with) an infectious disease, the infectious disease may be HIV, herpes, hepatitis or human papillomavirus. In some embodiments, the infectious disease is HIV. In some preferred embodiments, the infectious disease may be COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, a disease caused by SARS-CoV-2).

The methods of the present invention may include isolation and/or expansion steps. For example, the method can comprise isolating a population of NKT cells from the subject or from a sample derived from the subject. Optionally, the isolating step is at least 48 hours after administration of the glucocorticoid; 48 hours to 13 days after administration of glucocorticoids; Alternatively, it may be performed between 6 and 48 hours after administration of the glucocorticoid. In some embodiments (eg, embodiments in which the subject has cancer, an infectious disease or a microbial disease, or an autoimmune disease), isolating the NKT cells comprises administering the glucocorticoid within 3 hours after administration of the glucocorticoid, preferably administering the glucocorticoid This can be done within 1 hour. The sample may be selected from the group consisting of blood, plasma, tumor biopsy or surgically removed tumor, bone marrow, liver, spleen biopsy, and adipose or adipose tissue. In some embodiments, the method further comprises expanding the isolated NKT cells. In some embodiments, the method comprises activating the isolated NKT cells with an NKT cell activator. The NKT cell activator may include cytokines, chemokines, growth factors, and/or alpha GalCer (alpha-galactosylceramide; α-GalCer) sulfatide (3-O-sulfogalactosylceramide; SM4; galactosylsulfate). NKT modulators such as cerebroside).

The isolated NKT cells of the invention can be further engineered, for example, by transfecting the cells with a nucleic acid. Accordingly, in some embodiments, the method further comprises introducing a nucleic acid encoding the protein into the isolated NKT cell, and culturing the cell under conditions that promote expression of the protein. The protein may be one or more of T-cell receptor (TCR), chimeric antigen receptor (CAR), split, universal and programmable CAR (SUPRA-CAR). The CAR and/or TCR comprises an antigen binding domain that binds to an antigen selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA , ROR1, CAIX, Her2.

The NKT cells of the present invention find use in medicine. For example, the isolated NKT cells of the invention can be used medically, for example, in the treatment of cancer, autoimmune disease, or infectious disease (also called microbial disease) in a subject. In these embodiments, the method may comprise administering to a subject suffering from one of the aforementioned diseases a therapeutically effective amount of NKT cells isolated via the methods disclosed herein. In some embodiments, the subject to which the isolated NKT cells have been administered is the same subject from which the NKT cells have been isolated. Alternatively, the subject to which the isolated NKT cells have been administered is different from the subject from which the NKT cells have been isolated.

The NKT cells are administered to the subject by a method selected from the group consisting of: intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, intracerebrospinal fluid (CSF) injection, intratumoral direct injection, and solid tumor over or gel nearby.

The invention also extends to the use of glucocorticoids in the manufacture of a medicament for use in the methods of treatment disclosed herein.

The present invention further extends to the use of dexamethasone or other glucocorticoids to induce a population of NKT cells, wherein the population of NKT cells is induced by a method according to any one of statements 101-148.

The invention further extends to the use of dexamethasone or other glucocorticoids to recruit a population of NKT cells, wherein the population of NKT cells is recruited by a method according to any one of statements 101-148.

Also provided is an induced pluripotent stem cell derived from the NKT cell of the present invention. Accordingly, in one aspect, the present invention provides a method of producing an induced pluripotent stem cell (iPSC), the method comprising reprogramming the NKT cells isolated by the methods disclosed herein to produce an iPSC. The reprogramming may comprise introducing into the NKT cell one or more nucleic acids encoding Oct3/4, Klf4, Sox2, and C-myc. The nucleic acid may be a DNA (eg a DNA expression cassette) or an RNA molecule. The reprogramming may further comprise introducing into the NKT cell one or more expression cassettes encoding one or more of the following: Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog , and/or LIN28. The reprogramming may further comprise introducing a cassette of one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28 encoding mRNA into the NKT cell. . The iPSCs can then be induced to differentiate into, for example, NKT cells or into an NKT cell lineage.

The invention also provides an isolated population of natural killer T cells (NKT cells) or natural killer T cells (NKT cells) produced by the methods disclosed herein. In this regard, the NKT cells of the invention may be defined by their expression profile(s), which may be as described elsewhere herein. For example, the invention provides that a cell expresses CD3 and optionally expresses one or more of CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta; Provided is an isolated natural killer T cell (NKT cell) characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta. The isolated natural killer T cells (NKT cells) may be derived from a subject without disease.

NKT cells or precursors thereof may be isolated from a subject, wherein the NKT cells or precursors of NKT cells are in vivo prior to isolation or in vitro after isolation. a high dose glucocorticoid receptor (GR) modulator or ICAM3 modulator, wherein the CD3 expression level is at least 2-fold higher than the average level of CD3 expression in a reference NKT cell population from the subject. The NKT cells or precursors thereof may have been isolated from the subject, and the NKT cells or precursors of the NKT cells may be in vivo prior to isolation or in vitro after isolation. A high dose glucocorticoid receptor (GR) modulator or ICAM3 modulator, wherein the CD3 expression level is at least 2-fold higher than the average level of CD3 expression in a reference NKT cell population from a subject not contacted with the GR modulator or ICAM3 modulator.

The CD3 expression level of the isolated NKT cells and the population of reference NKT cells can be measured by any method known in the art, for example by flow cytometry. (The CD3 expression level of the isolated NKT cells and the CD3 expression level of the reference NKT cell population should both be measured using the same method.) When flow cytometry is used to measure the expression level of a marker such as CD3 , the equipment, reagents, and/or conditions described herein may be used in conjunction with any methods and protocols known in the art.

An isolated NKT cell of the invention may exhibit a CD3 expression level that is at least 3-fold, at least 4-fold, or at least 5-fold higher than the average level of CD3 expression in a reference NKT cell population. The population of reference NKT cells may have been obtained from the same subject prior to exposure to glucocorticoids.

The invention also provides an isolated population of natural killer T cells (NKT cells). An isolated population of NKT cells may be defined by their expression profile(s), which may be as described elsewhere. For example, an isolated population of natural killer T cells (NKT cells) may include at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD3, and/or CD4, CD8 , CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and TCR gamma/delta; It may be characterized as not expressing C-kit, B220, FoxP3, and/or TCR alpha/beta. In humans, the NKT cells may express CD56 in place of or in addition to CD49b. NKT cells may not express Sca1. Thus, NKT cells express CD3 and/or may express one or more of CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, Sca1, and TCR gamma/delta. The NKT cells express CD3 and/or may express one or more of CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, Sca1, and TCR gamma/delta. The NKT cells express CD3 and/or may express one or more of CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, and TCR gamma/delta. The NKT cells express CD3 and/or may express one or more of CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, and TCR gamma/delta. The present invention provides a glucocorticoid for use in a method of treating cancer, an autoimmune disease or an infectious disease (also referred to as a microbial disease) in a subject, the method comprising: about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone administering to the subject a glucocorticoid at a dose equivalent to For example, the present invention is used in a method for inducing tumor necrosis, inducing NKT tumor invasion, releasing immune activating cytokines, swallowing and killing tumor cells, promoting other immune cell infiltration into tumors and/or in a method for CD1d induced apoptosis in cancer patients. There is provided a glucocorticoid for: administering a glucocorticoid to a subject equivalent to about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone to induce a population of NKT cells of the invention as defined herein including administration in doses. For example, the present invention relates to a method for inducing tumor necrosis, inducing NKT cell tumor invasion, releasing immune activating cytokines, swallowing and killing tumor cells, promoting other immune cell infiltration into tumors and/or CD1d induced apoptosis in cancer patients. Provided is a glucocorticoid for use, the method comprising administering to a subject about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone and a glucocorticoid to recruit a population of NKT cells of the invention as defined herein; including administration in equivalent doses. For example, the present invention provides a glucocorticoid for use in a method of inducing virus death, inducing NKT recruitment, releasing immune activating cytokines, swallowing and killing virus-infected cells, and promoting other immune cell infiltration into virus-infected organs, said The method comprises administering to the subject a glucocorticoid at a dose equivalent to about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone to the subject to induce a population of NKT cells of the invention as defined herein. The HED of dexamethasone can take any value within the range of values disclosed herein.

Embodiments and experiments illustrating the principles of the present disclosure will now be discussed with reference to the accompanying drawings:
Figure 1 . Acute high-dose dexamethasone reduces mouse lymphocyte counts. As measured by whole blood count (cells/ul = absolute number obtained from CBC) after high-dose dexamethasone (18 mg/kg HED dexamethasone phosphate (DP)) 6 hours, 24 hours, 48 hours, 7 days, 13 days, and 21 days Absolute lymphocyte counts (ALC-NK and NKT cells) were significantly reduced compared to placebo. Near complete lymph node dissection was observed at 6 and 48 hours post-dose, an effect similar to that achieved with standard Cy/Flu chemotherapy (13 mg/kg HED cyclophosphamide and 0.8 mg/kg HED fludarabine).
2 . Acute high-dose dexamethasone reduces mouse B lymphocyte counts. B lymphocyte counts as measured by whole blood count (cells/ul = absolute number obtained from CBC) after high-dose dexamethasone (18 mg/kg HED DP) 6 hours, 24 hours, 48 hours, 7 days, 13 days, and 21 days decreased significantly compared to placebo. The lymphadenatic effect on B lymphocytes is similar to that achieved with standard Cy/Flu chemotherapy (13 mg/kg HED cyclophosphamide and 0.8 mg/kg HED fludarabine).
3 . Acute high-dose dexamethasone reduces mouse monocyte counts. After high-dose dexamethasone (18 mg/kg HED DP), monocyte counts as measured by whole blood counts (cells/ul = absolute number from CBC) were significantly reduced compared to placebo at 6, 24, and 48 hours. The ablation effect on monocytes is superior to that achieved by standard Cy/Flu chemotherapy (13 mg/kg HED cyclophosphamide and 0.8 mg/kg HED fludarabine).
Acute high-dose dexamethasone reduces mouse neutrophil counts. After high-dose dexamethasone (18 mg/kg HED DP), neutrophil counts as measured by whole blood counts (cells/ul = absolute number from CBC) were significantly reduced compared to placebo at 6, 24, and 48 hours.
Figure 5 . Acute high-dose dexamethasone preserves mouse platelets. Acute high-dose dexamethasone (18 mg/kg HED DP) had no effect on platelet count as measured by whole blood count (cells/ul = absolute number obtained from CBC). Thus, acute high-dose dexamethasone eliminates the need for blood transfusions and provides a safer, non-toxic alternative to chemotherapy.
6 . Acute high-dose dexamethasone preserves hematopoietic stem cells. The number of viable hematopoietic stem cells measured between 6 hours and 35 days in naive mice treated with placebo or acute high-dose dexamethasone is shown. Acute high-dose dexamethasone (18 mg/kg HED DP) does not significantly alter the number of live hematopoietic stem cells. Therefore, nonmyelodysplastic therapy, represented by acute high-dose dexamethasone, can eliminate the need for stem cell transfusion for hematopoietic recovery after immune reestablishment.
7 . Acute high-dose dexamethasone induces NKT upregulation ( FIG. 7 ) and generation of a new population of NKT cells (AVM-NKT). Total NKT cell counts as measured by whole blood counts (cells/ul = absolute number from CBC) were decreased compared to placebo at 6 and 24 hours after high-dose dexamethasone (18 X mg/kg HED DP). Surprisingly, the total number of NKT cells as measured by whole blood count increased 48 hours after high-dose dexamethasone, and then gradually decreased until about 13 days after high-dose dexamethasone treatment. With administration of standard Cy/Flu chemotherapy (13 mg/kg HED cyclophosphamide and 0.8 mg/kg HED fludarabine), no such increase in NKT cell count was observed 48 hours after treatment.
Figure 8 . After high-dose dexamethasone treatment, two NKT populations can be identified in the peripheral blood. Irradiation of peripheral blood by flow cytometry after acute high-dose dexamethasone identified two NKT cell populations: NKT cells (central rectangular gate) defined as CD3medCD49b+ (human CD56) corresponding to NKT cells previously described; and a novel population of NKT cells defined as CD3highCD49b+ (human CD56; AVM-NKT cells; center right rectangular gate). AVM-NKT cells are very bright for CD49b+ (human CD56) and CD3 compared to known NKT cells expressing CD3 with mean fluorescence intensity (MFI) 1/2 to 1 log lower than AVM-NKT.
Figure 9 . Time course of AVM-NKT upregulation. Quantification of AVM-NKT cells per microliter of blood using CBC and flow cytometry results. AVM-NKT cells are evident in the blood of naive mice between 48 hours and 13 days after a single high-dose dexamethasone (HED 18.1 mg/kg DP PO) treatment; ^* = Statistically significant.
Figure 10 . Changes in the A20 tumor environment induced by high-dose dexamethasone (HED 18 mg/kg DP) treatment. After 48 hours, an increase in necrosis was evident in the tumors of mice treated with high-dose dexamethasone compared to placebo.
11 . Acute high-dose dexamethasone (AVM0703; HED 18.1 mg/kg PO) significantly delayed the growth of A20 B-cell lymphoma compared to placebo. Days of high-dose dexamethasone or placebo administration are indicated by arrows.
12 . Time course of the percentage of typical NKT (non-AVMNKT; left) or AVM-NKT (right) cells in naive male C57Bl/6 mice after a single oral dose of 15 mg/kg of dexamethasone base as measured by flow cytometry that is cell CD4 positive. Bars represent the mean of each time point.
13 . Time course of the percentage of typical NKT (non-AVMNKT; left) or AVM-NKT (right) cells in naive male C57Bl/6 mice after a single oral dose of 15 mg/kg of dexamethasone base as measured by flow cytometry that was CD8 positive. Bars represent the mean of each time point.
14 . After a single oral dose of 15 mg/kg of dexamethasone base as measured by flow cytometry, CD4 CD8 double positive (top left), CD8 single positive (top right), CD4 single positive (bottom left), or CD4 CD8 double negative (bottom right). Time course of percentage of typical NKT (non-AVMNKT; left) or AVM-NKT (right) cells in naive male C57Bl/6 mice. Bars represent the mean of each time point.
15 . CD3 positive median fluorescence intensity (MFI) for typical NKT (left) or AVM-NKT (right) cells of naive male C57Bl/6 mice after a single oral dose of 15 mg/kg of dexamethasone base as measured by flow cytometry (top graph) and time course of arithmetic mean fluorescence intensity (bottom graph). Typical NKT cells are CD49b positive (human CD56) with an MFI equivalent to AVM-NKT cells. Bars represent the mean of each time point.
16 . CD4 positive median fluorescence intensity (MFI) for typical NKT (left) or AVM-NKT (right) cells of naive male C57Bl/6 mice after a single oral dose of 15 mg/kg of dexamethasone base as measured by flow cytometry (top graph) and time course of arithmetic mean fluorescence intensity (bottom graph). Typical NKT cells are CD49b positive (human CD56) with an MFI equivalent to AVM-NKT cells, but are CD3 positive with an MFI approximately 1 log lower than AVM-NKT cells. Bars represent the mean of each time point.
17 . CD8 positive median fluorescence intensity (MFI) for typical NKT (left) or AVM-NKT (right) cells of naive male C57Bl/6 mice after a single oral dose of 15 mg/kg of dexamethasone base as measured by flow cytometry (top graph) and time course of arithmetic mean fluorescence intensity (bottom graph). Typical NKT cells are CD49b positive (human CD56) with a cell-equivalent MFI, but are CD3 positive with an MFI about 1 log lower than AVM-NKT cells. Bars represent the mean of each time point.
18 . Mean fluorescence intensity expression of Ly6G on all CD45 faint and positive cells from placebo or 15 mg/kg HED dexamethasone base-treated mice 48 hours after administration was measured by flow cytometry (MacsQuant, Miltenyi). Dexamethasone treated mice have a population of CD45 faint, positive cells expressing Ly6G at a much higher level (MFI about 104) than the majority of CD45 positive cells (MFI about 103). 104 MFI Ly6G positive cells from dexamethasone treated mice also express very high CD3 (about 1 log higher MFI than T lymphocytes and other NKT cells) and are also CD49b positive (human CD56).
19 . AVM_NKT expresses Ly6G for antitumor, antiviral and antibacterial responses. The scatter plot shows the CD3 fluorescence intensity on the X-axis versus the Ly6G fluorescence intensity on the Y-axis for all CD45 positive cells 48 hours after AVM0703 HED 18.1 mg/kg dose. AVM_NKT is highlighted in black. Placebo CD3 vs. Ly6G scatter overlaps within the area surrounded by black outlines for comparison.
Figure 20. After treatment with high-dose dexamethasone, a new population of T cells with very high CD3 in peripheral blood can be identified. Peripheral blood examination by flow cytometry after acute high-dose dexamethasone identified two NKT cell populations: NKT cells defined as CD3medCD49b+ corresponding to previously described NKT cells (central rectangular gate); A novel population of NKT cells defined as CD3highCD49b+ (AVM-NKT cells; center-right rectangular gate), as well as novel very high CD3 T cells (black circles).
Figure 21. High-dose dexamethasone induces/activates/mobilizes a new population of dendritic cells with very high CD11b. Dendritic cells with very high CD11b express CD11b approximately 1 log higher than conventional CD11b+ dendritic cells. High-dose dexamethasone also increases the number of pre-existing CD11b+ dendritic cells.
22. AVM0703 (18 mg/kg HED dexamethasone phosphate (DP)) delays A20 B-lymphoma growth in a mouse model. Group mean tumor volumes are shown during the course of the study. Arrows indicate the date of administration to mice. The graph shows a clear separation of placebo (n = 4) and AVM0703-treated (n = 5) mice over the course of the study.
23. AVM0703 delays endpoint and eradicates A20 tumor cells in a mouse lymphoma model. (A) Image is a 2X bright field microscopy image showing that tumor growth in AVM0703-treated mice was pseudogrowth and tumors in AVM0703-treated mice were mostly necrotic, and tumor cells were not evident even in areas without complete necrosis and thus not true tumor growth. to be. (B) Endpoint curves for study mice where the x-axis is one post-inoculation. The median time to endpoint for placebo-treated mice was 22 days and the median time to endpoint for AVM-treated mice was 41 days. Kaplan-Meier analysis determined that the time to endpoint of AVM treatment was significantly longer ( ^** p<0.01). (C) Brightfield image of a thick-sectioned tumor. Macroscopic examination of the tumor revealed differences between placebo and AVM0703 treated mice. Therefore, the tumors were cut thickly and examined microscopically in bright field. Tumors in placebo-treated mice (left) were highly cellular with small areas of necrosis, whereas tumors in AVM0703-treated mice (right) were mostly necrotic and acellular.
24. Repeat AVM0703 (18 mg/kg HED dexamethasone phosphate (DP)) administration, up to 7 administrations, does not reduce body weight. Mean body weight graphs for each group of mice (n = 4 placebo, n = 5 AVM0703) over the course of the study. Arrows below the x-axis indicate dosing days. The dotted line represents a loss of 20% of the mean body weight of all mice at the start of the study. One mouse reached the threshold after 8 doses and was euthanized.
25. Organ weight to body weight ratio at study endpoints. Graph of organ weight to body weight ratio. Colon weight was significantly higher in AVM0703-treated mice (n = 5) compared to placebo (n = 4); However, this may be due to a significant increase in the age of the AVM0703-treated mice at the time of euthanasia (14-40 days older than the placebo mice at the time of euthanasia). ^* p<0.1.
Figure 26. Duration of responsiveness to repeat AVM0703 as determined by splenic and thymus weight loss BALB/c mice were randomized into two groups and orally with 18.06 mg/kg AVM0703 HED DP (n = 5) or placebo (n = 4). dealt with Graphs of body weight versus spleen (left) and thymus (right) weight ratios based on the number of days since the last dose of AVM0703. The number near each dot represents the total number of AVM0703 doses the mice received before reaching the study endpoint. The dotted line represents the mean spleen or thymus ratio to body weight after placebo treatment. AVM0703 continued to affect both the thymus and spleen until up to 7 doses, and spleen and thymus weight decreased relative to placebo on days 1 and 3 post-dose and nearly returned to placebo values by day 6 after 7 doses. AVM0703 Spleen and thymus weight loss appears to be lost after 8 doses. Avg. = average; DP = dexamethasone phosphate; HED = human equivalent dose.
27. Tumors stained with hematoxylin and eosin and imaged at 20X magnification. Stars indicate areas of necrosis. The black arrow indicates the tumor growth area extending in the direction of the arrow. A. 0 mg/kg DP; B. 7 mg/kg DP; C. 18 mg/kg DP; E. 25 mg/kg DP; Red areas indicate bleeding; E. Mean pathology score averaged across tumors where n = 2 tumors, DP = dexamethasone phosphate.
28. Tumor CD3 expression. Images of tumors stained via immunohistochemistry for CD3 and imaged at 100X magnification. The black arrow indicates the infiltration of CD3+ circular cells in the direction of the arrow. 'N' represents the tumor growth region. A.0 mg/kg DP, 'BV' represents blood vessels; B. 7 mg/kg DP; C. 18 mg/kg DP; D. 25 mg/kg DP. DP = dexamethasone phosphate.
29. Tumor NKp46 expression. Images of tumors stained via immunohistochemistry for the NK cell marker NKp46 and imaged at 100X magnification. Stars indicate areas of necrosis. Black arrows indicate examples of cells positive for NKp46. 'N' represents the tumor growth region. A. 0 mg/kg DP; B. 7 mg/kg DP; C. 18 mg/kg DP; D. 25 mg/kg DP. DP = dexamethasone phosphate; NK = natural killing.
30. Tumor CD49b expression. Images of tumors stained via immunohistochemical staining for CD49b and imaged at 100X magnification. Black arrows indicate examples of cells positive for CD49b. Blue arrows indicate vascular or endothelial cells labeled with CD49b. 'N' represents the tumor growth region. A. 0 mg/kg DP; B. 7 mg/kg DP; C. 18 mg/kg DP; D. 25 mg/kg DP. DP = dexamethasone phosphate.
Figure 31. Tumor apoptosis. Images of tumors stained via immunohistochemistry for the apoptosis marker caspase 3 and imaged at 40X magnification. Stars indicate areas of necrosis. Black arrows indicate examples of cells that are positive for caspase 3. 'N' represents the tumor growth region. A. 0 mg/kg DP; B. 7 mg/kg DP; C. 18 mg/kg DP; D. 25 mg/kg DP. DP = dexamethasone phosphate.
Figure 32. Resorbed tumors in mice treated with AVM0703 and Cy/Flu combination.
Figure 33. Tumor Examples of Study AVM_CANMOD_05 - Lymphocyte Depleted Subset. Left: Example of a placebo tumor; 956 mm ³ , L 15.06, W 11.27 mm, 0.54 g; Right: AVM0703 (25 mg/kg) tumor example; 203.25 mm ³ , L 7.67, W 7.28 mm, 0.086 g.
Figure 34. Tumor Examples of Study AVM_CANMOD_05 - Endpoint Analysis Subset. Left: Example of a placebo tumor. Right: 18 mg/kg mouse 11 AVM0703 tumor example.
Figure 35. Evidence of oncolytic syndrome in CCRF CEM tumor bearing mice treated with AVM0703. Oncolysis is indicated by significant green, oily areas within the tumor.
Figure 36. Tumor volume graph of mice seeded with CCRF-CEM cells and treated with placebo (n = 2) or 18 mg/kg AVM0703 (n = 3). Mice were administered once a week starting on day 7 after inoculation, and dosing events were indicated by black arrows. Mice were euthanized if the tumor volume exceeded 1500 mm ³ .
Figure 37. Endpoint curve of mice inoculated with CCRF-CEM cells. Mice were euthanized when tumor volume reached the endpoint at 1500 mm ³ . Both the placebo and AVM0703 treatment groups were n=2.
38. AVM0703 induces long-term immunity against human T-ALL xenografts in NCR nude mice. AVM0703-treated mice were rechallenged with human T-ALL (CCRF-CEM cell line) on day 118 and no tumor growth was observed until day 164, indicating that AVM0703 induces long-term immunity.
Figure 39. 3-6 mg/kg DSP CD45/CD56 scattergrams of treated osteoarthritis patients. AVM-NKT cells (indicated by square boxes) were identified and CD45 dark and CD56 very bright (CD49b in mice) as in mice.
Figure 40. Flow cytometry data of healthy donors and prostate cancer patients 1 and 3 hours after administration of 6 mg/kg AVM0703. A novel CD45dim CD56bright cell population (circles) in prostate cancer patients is evident 1 hour after injection. These data indicate that human patients recruit cells corresponding to the identified AVM-NKT cells in mice.

The present disclosure relates to: a method of producing/activating/mobilizing a population of natural killer T cells (NKT cells), an isolated NKT cell or an isolated population of NKT cells produced by the method, and wherein the NKT cells are a method of treatment induced in or administered to a subject; methods of producing/activating/mobilizing a population of T cells, T cells or isolated populations of T cells isolated by such methods, and methods of treatment wherein the T cells are induced in or administered to a subject; methods of producing/activating/mobilizing populations of dendritic cells, isolated dendritic cells or isolated populations of dendritic cells produced by such methods, and methods of treatment wherein the dendritic cells are induced in or administered to a subject; and methods of activating a population of dendritic cells, dendritic cells or isolated populations of dendritic cells isolated by such methods, and methods of treatment wherein the dendritic cells are induced in or administered to the subject.

In some embodiments, the disclosed methods are methods of generating natural killer T cells (NKT cells) and a population of T cells, and activating the population of dendritic cells. In another embodiment, the disclosed method is a method of recruiting a population of natural killer T cells (NKT cells), T cells, and/or dendritic cells. As used herein, “mobilizing” these cells refers to promoting their migration from lymphoid organs/tissues (eg, thymus and spleen) to the systemic circulation (then other sites, eg, tumor sites). can mean The disclosed methods may include many of the above aspects. For example, the methods of the present disclosure can induce generation of a NKT cell population as described herein and recruit a NKT cell population as described herein. For example, the methods of the present disclosure can induce the production of a NKT cell population as described herein in the thymus and/or spleen and/or bone marrow, and in the thymus and/or spleen and/or bone marrow as described herein. The same NKT cell population can be recruited.

As disclosed herein, a method of generating a population of natural killer T cells (NKT cells), a method of generating a population of T cells, and/or a method of generating or activating a population of dendritic cells is a method of generating a glucocorticoid receptor ( GR) a modulator or an ICAM3 modulator. A glucocorticoid receptor (GR) modulator or ICAM3 modulator induces a population of NKT cells, induces a population of T cells, and/or activates a population of dendritic cells in a subject. A glucocorticoid receptor (GR) modulator or ICAM3 modulator is capable of recruiting a population of NKT cells, recruiting a population of T cells, and/or activating or recruiting a dendritic population in a subject.

Also disclosed are isolated populations of NKT cells, isolated NKT cells, isolated populations of T cells, isolated T cells, isolated populations of dendritic cells, and isolated dendritic cells that can be produced by the disclosed methods.

Disclosed NKT cells can be characterized by the pattern of surface proteins they express. In some embodiments, the disclosed NKT cells are capable of expressing CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. In some embodiments, the disclosed NKT cells may not express C-kit, B220, FoxP3, and/or TCR alpha/beta. In humans, NKT cells may express CD56 in place of or in addition to CD49b. In some embodiments the NKT cells do not express Sca1. Thus, the disclosed NKT cells are capable of expressing CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. The disclosed NKT cells may express CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. The disclosed NKT cells may express CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. The disclosed NKT cells may express CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. In some embodiments, the disclosed NKT cells may or may not express CD44, CD69, and/or CD25. In some embodiments the disclosed NKT cells are capable of expressing CD56.

In embodiments related to the disclosed population of NKT cells, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, It may be characterized as expressing CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. NKT cells can express CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. NKT cells express CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. NKT cells may express CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. NKT cells express CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. In some such embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are C-kit, B220, FoxP3, and/or TCR alpha/beta. It can be characterized in that it does not express. In some embodiments the population of NKT cells can be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells express CD56.

Initiated T cells can be characterized by the pattern of surface proteins they express. Initiated T cells express CD3 with very high MFI. In embodiments related to the disclosed population of T cells, the population of T cells can be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells express CD3. . In some embodiments, the disclosed T cells are capable of expressing CD3, CD4, CD45, and/or CD49b (human CD56). In some embodiments, the disclosed T cells are capable of expressing TCR gamma/delta. In some embodiments, the disclosed T cells are capable of expressing TCR alpha/beta. In some embodiments, the disclosed T cells are capable of expressing CD8. In some embodiments, the disclosed T cells may not express CD8. In embodiments related to the disclosed population of T cells, the population of T cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD45, and/or CD49b (human CD56). In some embodiments related to the disclosed population of T cells, the population of T cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells express TCR gamma/delta. can be done with In some embodiments related to the disclosed population of T cells, the population of T cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells express TCR alpha/beta. can be done with In some such embodiments, the population of T cells can be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells express CD8. In some such embodiments, the population of T cells can be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells express CD8. In a preferred embodiment the T cell or population of T cells expresses CD8 and/or TCR gamma/delta.

The disclosed dendritic cells can be characterized by the pattern of surface proteins they express. The disclosed dendritic cells express CD11b. In embodiments related to the disclosed population of dendritic cells, the population of dendritic cells can be characterized by at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the dendritic cells expressing CD11b. .

Expression of surface proteins in cells can be readily determined using techniques well known to those skilled in the art—eg, enzyme linked immunosorbent assay (ELISA), magnetically activated cell sorting (MACS), or flow cytometry techniques. Flow cytometry uses the properties of light scattered from cells bound by fluorescently tagged antibodies to identify cells expressing a surface protein of interest. Flow cytometry can determine whether a cell expresses a protein of interest, as well as indicate the amount of protein expressed by the cell based on fluorescence intensity. In flow cytometry readouts, and as used herein: “+” (or “positive”) indicates expression of a given surface protein; "-" (or "negative") indicates no expression of the given surface protein; and "+/-" indicates bimodal expression of a given surface protein. Expressions such as "light" (sometimes "high" or "++"), "dark" (sometimes "low"), and "normal" are used to indicate the relative amount of a particular cell surface protein.

CD3

CD3 (Cluster of Differentiation 3) is a T cell co-receptor that helps activate cytotoxic T cells (CD8+ naive T cells) and T helper cells (CD4+ naive T cells). Because CD3 is required for T cell activation, drugs targeting it (eg monoclonal antibodies) have been investigated as immunosuppressive therapies (eg otelixizumab) for type 1 diabetes and other autoimmune diseases. have. The known NKT cells described in the literature express CD3 with a mean fluorescence intensity (MFI) about 1 log lower than the NKT cells of the present disclosure. Similarly, the known T cells and NKT cells described in the literature express CD3 with a mean fluorescence intensity (MFI) about 1-1.5 log lower than the T cells of the present disclosure.

In some embodiments, the NKT cells of the present disclosure express CD3. In some embodiments, the NKT cells of the present disclosure are CD3+/very bright. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3 can be expressed. In some embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells can be CD3+/very bright.

T cells of the present disclosure are CD3+/very bright. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are Can be CD3+/very bright.

CD4

CD4 (Cluster of Differentiation 4) is a glycoprotein found on the surface of immune cells, including T-helper cells and monocytes. CD4 is a co-receptor of the T cell receptor (TCR), which helps to communicate with antigen presenting cells for antigen-induced T cell activation. Cross-linking of CD4 can induce T cell apoptosis via the Fas ligand pathway.

In some embodiments, the NKT cells of the present disclosure express CD4. In some embodiments, the NKT cells of the present disclosure are CD4+/very bright. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD4 can be expressed. In some embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells can be CD4+/very bright.

In some embodiments, the T cells of the present disclosure express CD4. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD4 can be expressed.

CD8

CD8 (Cluster of Differentiation 8) is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR). It is mainly expressed on the surface of cytotoxic T cells, but is also expressed on natural killer cells. It plays a role in T cell-antigen interactions and T cell signaling in T cells.

In some embodiments, the NKT cells of the present disclosure express CD8. In some embodiments, the NKT cells of the present disclosure are CD8+/dark. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD8 can be expressed. In some embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells may be CD8+/dark. In some embodiments, the disclosed NKT cells may not express CD8.

In some preferred embodiments, the NKT cells of the present disclosure express CD4 and CD8. In some preferred embodiments related to the population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% NKT Cells can express CD4 and CD8. In some embodiments the NKT cells of the disclosure are not CD4 and CD8 double negative. In some embodiments related to the population of NKT cells of the present disclosure, none of the NKT cells are CD4 and CD8 double negative.

In some embodiments, T cells of the present disclosure may not express CD8. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD8 may not be expressed.

CD45

CD45 (Cluster of differentiation 45; protein tyrosine phosphatase, receptor type; also known as PTPRC) is an essential regulator of T-cell and B-cell antigen receptor signaling, and a marker of all leukocytes. CD45 expression is essential for T cell activation by TCR. CD45 may be a receptor for CD26.

In some embodiments, the NKT cells of the present disclosure express CD45. In some embodiments, the NKT cells of the present disclosure are CD45+/dark. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD45 can be expressed. In some embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells may be CD45+/dark.

In some embodiments, T cells of the present disclosure are capable of expressing CD45. In some embodiments, the T cells of the present disclosure are CD45+/dark. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD45 can be expressed. In some embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of T cells may be CD45+/dark.

CD45 may be an isoform of CD45, such as CD45RA, CD45RO and/or CD45RABC (also known as CD45R; also known as B220).

CD49b

CD49b (Cluster of Differentiation 49b; also known as Integrin Alpha-2) is an integrin alpha subunit. It constitutes half of the α2β1 integrin duplex. CD49b is used as a marker of natural killer (NK) cells; It is known that the cytotoxicity of NK cells expressing CD49b is much greater than that of NK cells not expressing CD49b.

In some embodiments, the NKT cells of the present disclosure express CD49b. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD49b can be expressed.

In some embodiments, the T cells of the present disclosure are capable of expressing CD49b. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD49b can be expressed.

CD56

CD56 (Cluster of Differentiation 56; also known as Neuronal Adhesion Molecule, NCAM) is a homologous binding glycoprotein expressed on the surface of neurons, glial and skeletal muscles. CD56 expression is associated with natural killer cells.

In some embodiments, the NKT cells of the present disclosure express CD56. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD56 can be expressed.

In some embodiments, the NKT cells of the present disclosure are CD56+/bright. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are It can be CD56+/bright.

CD62L

CD62L (Cluster of Differentiation 62L; also known as L-selectin) is a marker of cell activation. CD62L, also referred to as L-selectin, is capable of mediating cell-cell adhesion, which initiates the process by which cells cross the endothelium and migrate from the blood to tissues and organs.

In some embodiments, the NKT cells of the present disclosure express CD62L. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD62L can be expressed.

NK1.1

NK1.1 (Killer Cell Lectin-Like Receptor Subfamily B, Member 1; KLRB1; NKR-P1A; also known as CD161 (Cluster of Differentiation 161)) is a marker of mature NK cells and its activation is otherwise insensitive to NK cells. It can induce the killing of one target and also induce the proliferation of NK cells. NKT cells were first observed as a population of T lymphocytes expressing this pan-NK cell marker.

In some embodiments, the NKT cells of the present disclosure express NK1.1. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are It can express NK1.1.

Ly6G

Ly6G (lymphocyte antigen 6 complex locus G6D) is a marker for fully mature and differentiated neutrophils or granulocytes and is also associated with antitumor responses. Ly6G is generally a marker for monocytes and neutrophils and granulocytes, which distinguishes the NKT cells of the present disclosure from known NKT cells and is capable of directly killing cancer cells expressing CD1d, as well as other NK cells and B and T cells. This indicates that they can secrete lymphocytes and cytokines, but can also directly swallow cancer cells and pathogens.

In some embodiments, the NKT cells of the present disclosure express Ly6G. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are Ly6G can be expressed.

CD1

The CD1 molecule is a lipid presenting glycoprotein. Humans express five CD1 proteins (CD1a-e), of which four (CD1a-d) can migrate to the cell surface and display lipid antigens on T cell receptors. This interaction can benefit both non-cognate and cognate T cells to B cells, the latter eliciting an anti-lipid antibody response. All CD1 proteins are capable of binding a wide range of structurally different exogenous and endogenous lipids, but each favors one or more lipid classes (Kaczmarek et al, 2017, incorporated herein by reference in its entirety). This unorthodox binding behavior is the result of the sophisticated architecture of the CD1 binding cleft and unique intracellular trafficking pathways. Taken together, these features make the CD1 system a versatile player in immune responses at the intersection of innate and adaptive immunity. The CD1 system may be involved in numerous infectious, inflammatory and autoimmune diseases, but its involvement may lead to opposite consequences depending on various pathologies (Kaczmarek et al , 2017).

CD11b

CD11b (differentiation cluster molecule 11b, CR3a and integrin alpha M, also known as ITGAM) is a member of the integrin family that pairs with CD18 to form a CR3 heterodimer. CD11b is expressed on the surface of many leukocytes, including monocytes, neutrophils, natural killer cells, granulocytes and macrophages. The known dendritic cells described in the literature express CD11b with a mean fluorescence intensity (MFI) about one load lower than the dendritic cells of the present disclosure. Dendritic cells of the present disclosure are CD11b+/very bright. In embodiments related to the population of dendritic cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the dendritic cells are CD11b+/very bright.

major histocompatibility complex; MHC

MHC was discovered in 1936 by Gorer and Snell et al . Skin transplantation experiments in mice revealed that self-awareness and non-self-awareness depended on genetic background. Snell et al named a group of mouse genes that determine self/nonself as histocompatibility-2 (H-2). The genomic loci of MHC encode polymorphic cell membrane-bound glycoproteins (antigens) known as MHC classic class I and class II molecules, which modulate immune responses by presenting peptides of fragmented proteins to circulating cytotoxic and helper T lymphocytes, respectively. do. Classical MHC class 1 proteins are subdivided into HLA-A, HLA-B and HLA-C (Nakamura et al, 2019, incorporated herein by reference in its entirety). Conversely, HLA-E, HLA-F, HLA-G, MHC class I polypeptide related sequence A (MICA) and FcRn, etc. are classified as non-classical MHC class I.

Classical class I molecules of MHC are expressed in most tissues and are intracellularly directed against T-cell receptors on specific CD8+ T cells to non-covalently bind b2-microglobulins to induce their activation and/or cytotoxicity. peptide antigens (which are 8-11 amino acids in length) processed in (Shiina et al 2016, incorporated herein by reference in its entirety). Treated peptides can arise from the cell's own proteome or from foreign intracellular pathogens. Mature dendritic cells present peptides derived from the captured antigen by endocytosis using the MHC class I system. This process, called cross-presentation, plays an important role in initiating the response of specific T CD8+ lymphocytes in peripheral lymphoid organs (Shiina et al ). In addition, MHC classical class I proteins are ligands for killer-cell immunoglobulin-like receptors that modulate the cytotoxic activity of cytotoxic T cells and natural killer cells and leukocyte immunoglobulin-like receptors expressed on myelomonocytes and other leukocyte lineages. can act as In contrast to classical class I antigens, classical class II antigens form heterodimeric structures specialized for the presentation of exogenous peptides (15-25 amino acids in length) on the surface of lymphatic cells to CD4+ helper T lymphocytes of the immune system. Class II gene expression is mainly restricted to lymphocytes such as B cells, monocytes, macrophages, epithelial cells, dendritic cells and activated T cells. MHC class II proteins were identified as HLA-DR, HLA-DP and HLA-DQ. MHC class II genes are HLA-DRA1, HLA-DQA1, HLA-DPA1 encoding α chain, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5 (HLA-DRB3/4/5), HLA-DQB1, and It contains the HLA-DPB1 encoding β chain. HLA-DRA1 forms a heterodimer with HLA-DRB1 or HLA-DRB3/4/5 (Nakamura et al ). Similarly, HLA-DQA1 and HLA-DPA1 are also associated with HLA-DQB1 and HLA-DPB1, respectively. HLA-DRs are divided into five groups according to the antigenic group consisting of DR1, DR51, DR52, DR53 and DR8. Both the DR1 and DR8 groups consist of only DRB1 as expressed genes. In contrast, the DR51, DR52, and DR53 groups consist of genes that contain DRB1 in common and also expressed DRB5, DRB3, and DRB4, respectively, which are thought to have been generated by gene duplication from the DRB1 gene (Nakamura et al ).

Both classical class I and class II genes are often highly polymorphic to preserve interindividual variability in antigen presentation ability and to help species survive and defend natural selection pressures from a variety of infectious agents. Non-classical class I and class II antigens are similar in structure to their classical class I or class II counterparts, but are generally much less polymorphic, and have variable or limited tissue expression and function that differ markedly from classical class I or class II antigens. Moreover, several non-classical MHC class I genes are located outside the MHC (Shiina et al ).

The loci of the HLA complex (eg HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP) are highly polymorphic, resulting in very large combinations (haplotypes). However, MHC exhibits strong linkage disequilibrium, indicating non-random association of alleles at multiple loci. This linkage disequilibrium in the MHC region often results in specific combinations for each position in the MHC. When two genetic polymorphisms are present on the same chromosome, the two polymorphisms are classified as linked (Nakamura et al ). Given that genetic recombination occurred in a biologically customary manner, polymorphisms at different sites cannot be determined as in the linked state. However, linkage disequilibrium is a condition in which a specific gene polymorphism can be predicted with a very high probability based on polymorphism information in a distant region. In MHC, loci are concentrated in a narrow region on chromosome 6, so recombination between each gene is less likely. Thus, genes such as HLA-A, HLA-B, HLA-C, and HLA-DRB1 are often inherited in linkage disequilibrium conditions. As the HLA gene polymorphism analysis progresses, haplotypes associated with certain diseases frequently found in certain species are being revealed. Haplotypes specific to these ethnic groups are thought to be involved in the formation of ethnic groups. Therefore, these haplotypes are commonly used to find ethnic roots.

In humans, MHC classical class I genes are critically involved in organ transplant rejection and graft-versus-host disease after hematopoietic stem cell transplantation. Various associations have been demonstrated between HLA class I molecules and numerous autoimmune diseases, as well as infectious diseases and adverse drug reactions. In addition to their essential role in refining the adaptive immune response, the role of MHC class I genes has been demonstrated at various stages of reproduction, such as pregnancy maintenance, mate selection, and kin recognition. MHC was also regarded as a system that avoids inbreeding and primarily sexually selects due to histocompatibility to play a secondary role. MHC class I gene products also influence central nervous system development and plasticity, neuronal interactions, synaptic function and behavior, cerebral hemisphere specialization, and neurological and psychiatric disorders. Therefore, the human MHC class I region is one of the most biomedically diverse and important genomic regions (Shiina et al ).

TCR Gamma/Delta

T-cell receptor gamma delta (TCR gamma/delta; TCR γδ) is a T-cell receptor consisting of one γ (gamma) chain and one δ (delta) chain. TCR gamma/delta expressing T-cells (gamma delta T cells) are important recognizers of lipid antigens expressed on cancer cells as well as stress cells such as cancer cells, microbial and virally infected cells and autoreactive lymphocytes. Gamma delta T cells have a more evolutionarily primitive innate immune system that allows for a rapid beneficial response to a variety of external factors and a slow but highly antigenic B cell and T cell leading to long-lasting memory for subsequent challenge by the same antigen. It exhibits several properties that lie at the border between the adaptive immune systems that mediate specific immune responses. Gamma delta T cells can be considered a component of adaptive immunity in that they can rearrange the TCR gene to generate splicing diversity and develop a memory phenotype.

The most common human gamma delta variant is the blood Vgamma9/Vdelta2 variant, and Vdelta1-type gamma delta T cells in tumors are associated with prognosis. Vdelta3 variants have also been described as having Vdelta2 negative variants after CMV infection that reduce cancer risk. In contrast to MHC-restricted alpha beta T cells, gamma delta T cells can recognize MHC class Ib but do not require antigen processing and MHC presentation of peptide epitopes. Consequently, tumor cells downregulate MHC and gamma delta T cells to evade detection and thus also have the same potential to kill tumors with a low mutation load and are less affected by resistance issues. Gamma delta T cell tumor infiltration also showed the highest correlation with survival, and the incidence of graft-versus-host disease was low. Gamma delta T cells are naturally localized in various tissues for tumor detection and are preferred for homeopathic over alpha beta T cells.

In some embodiments, the NKT cells of the present disclosure express TCR gamma/delta. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are TCR gamma/delta can be expressed.

In some preferred embodiments, the NKT cells of the present disclosure express Ly6G and TCR gamma/delta. In some preferred embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT associated with the population of NKT cells of the present disclosure Cells can express Ly6G and TCR gamma/delta. Expression of both Ly6G and TCR gamma delta directly suggests that the NKT cells of the present disclosure are capable of engulfing cancer cells or pathogens in addition to the known functions of NKT cells.

In some embodiments, T cells of the present disclosure are capable of expressing TCR gamma/delta. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are TCR gamma/delta can be expressed.

Sca1

Sca1 (stem cell antigen-1; also known as Ly6A), along with other markers, is a common biological marker used to identify hematopoietic stem cells (HSCs). Sca-1 plays a role in hematopoietic progenitor/stem cell lineage fate and C-kit expression. Bright expression on the NKT cells of the present disclosure may indicate that they are activated memory stem cells.

In some embodiments, the NKT cells of the present disclosure express Sca1. In some embodiments, the NKT cells of the present disclosure are Sca1+/very bright. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are It can express Sca1. In some embodiments at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells can be Sca1+/very bright.

C-kit

C-kit (tyrosine-protein kinase KIT; CD117 (differentiation cluster 117); also known as mast/stem cell growth factor receptor (SCFR)) is a receptor tyrosine kinase protein expressed on the surface of hematopoietic stem cells. The fact that the NKT cells of the present disclosure do not express C-kit indicates that they are not hematopoietic stem cells.

In some embodiments, the NKT cells of the present disclosure may not express C-kit. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are C-kit may not be expressed.

B220

B220 (which is an isoform of CD45) is a marker for B cells.

In some embodiments, the NKT cells of the present disclosure may not express B220. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are may not express B220.

FoxP3

FoxP3 (forkhead box P3; also known as scuffin) is a member of the forkhead box protein family and is believed to function as a key regulator of regulatory pathways in the development and function of regulatory T cells. The fact that the NKT cells of the present disclosure do not express FoxP3 indicates that they are not regulatory cells and should not attenuate the immune response to cancer or pathogens.

In some embodiments, the NKT cells of the present disclosure may not express FoxP3. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are It may not express FoxP3.

TCR Alpha/Beta

T-cell receptor alpha beta (TCR alpha/beta; TCR αβ) is a predominantly TCR heterodimer consisting of one α (alpha) chain and one β (beta) chain.

In some embodiments, the NKT cells of the present disclosure may not express TCR alpha/beta. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are may not express TCR alpha/beta.

In some embodiments, T cells of the present disclosure are capable of expressing TCR alpha/beta. In embodiments related to a population of T cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are TCR alpha/beta can be expressed.

CD25

CD25 (Cluster of Differentiation 25; also known as interleukin-2 receptor alpha chain) is a transmembrane protein present on activated T and B cells and a marker of cell activation.

In some embodiments, the NKT cells of the present disclosure may be CD25+/-. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD25+/-.

CD44

CD44 (Cluster of Differentiation 44) is a cell surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is a receptor for hyaluronic acid and is involved in lymphocyte activation, lymphocyte return and recycling. CD44 expression is an indicator marker for effector-memory T cells - a subset of infection and cancer fighting T cells. Memory T cells have been “experienced” by encountering antigens during previous infections, cancer development, or previous vaccinations.

In some embodiments, the NKT cells of the present disclosure may be CD44+/-. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD44+/-.

CD69

CD69 (Cluster of Differentiation 69) is a human transmembrane type C lectin protein and is an early marker of cell activation. It is expressed in hematopoietic stem cells, T cells and several other immune cell types. CD69 induces NKT proliferation and can also activate other cells such as NK cells and lymphocytes.

In some embodiments, the NKT cells of the present disclosure may be CD69+/-. In embodiments related to a population of NKT cells of the present disclosure, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD69+/-.

In some embodiments, the NKT cells of the present disclosure are capable of expressing CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. In some embodiments, the NKT cells of the present disclosure are capable of expressing CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. In some embodiments, the NKT cells of the present disclosure are capable of expressing CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. In some embodiments, the NKT cells of the present disclosure are capable of expressing CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. In some embodiments, the NKT cells of the present disclosure are capable of expressing CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. In some preferred embodiments, the NKT cells of the present disclosure express CD4 and CD8. In some preferred embodiments, the NKT cells of the present disclosure express CD3, CD4, CD8, and CD49b. In some preferred embodiments, the NKT cells of the present disclosure express CD3, CD4, CD8, and CD56. In some preferred embodiments, the NKT cells of the present disclosure express Ly6G and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD49b, Ly6G, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD56, Ly6G, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD4, CD8, CD49b, Ly6G, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD4, CD8, CD56, Ly6G, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, Sca1, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, Sca1, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, and TCR gamma/delta. In some preferred embodiments the NKT cells of the present disclosure express CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, and TCR gamma/delta.

In some particularly preferred embodiments the NKT cells of the present disclosure express CD3, CD45, and/or CD56. In some such embodiments, the NKT cells of the disclosure are CD3+/bright or CD3+/very bright, and/or CD45+/dark, and/or CD56+.

In some embodiments, the NKT cells of the present disclosure may not express C-kit, B220, FoxP3, and/or TCR alpha/beta. In some embodiments, the NKT cells of the disclosure do not express C-kit, B220, FoxP3, or TCR alpha/beta. In some preferred embodiments, the NKT cells of the present disclosure express CD4 and CD8 and do not express C-kit, B220, FoxP3, and/or TCR alpha/beta. In some preferred embodiments, the NKT cells of the present disclosure express Ly6G and TCR gamma/delta and do not express C-kit, B220, FoxP3, and/or TCR alpha/beta.

In some embodiments, the NKT cells of the disclosure are CD44+/-, CD69+/-, and/or CD25+/-. In some embodiments, the NKT cells of the disclosure are CD44+/-, CD69+/-, and CD25+/-.

In embodiments related to the population of NKT cells of the present disclosure, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45 , CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta. In some such embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are C-kit, B220, FoxP3, and/or TCR alpha/beta. It may be characterized in that it does not express. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, It may be characterized as expressing Ly6G, Sca1, and/or TCR gamma/delta. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, It may be characterized as expressing Ly6G, Sca1, and/or TCR gamma/delta. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta. In some embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1, Ly6G, and/or TCR gamma/delta.

In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD56 CD62L, NK1. 1, Ly6G, Sca1, and TCR may be characterized as expressing gamma/delta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1 , Ly6G, Sca1, and TCR gamma/delta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD56 CD62L, NK1.1, It can be characterized as expressing Ly6G, Sca1, and TCR gamma/delta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1 , Ly6G, and TCR gamma/delta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD56 CD62L, NK1.1, It can be characterized as expressing Ly6G, and TCR gamma/delta. In some such embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells expressing C-kit, B220, FoxP3, or TCR alpha/beta. It can be characterized as not doing. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1 It can be characterized as expressing .1, Ly6G, Sca1, and TCR gamma/delta and not expressing C-kit, B220, FoxP3, or TCR alpha/beta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1 , Ly6G, Sca1, and TCR gamma/delta, and not C-kit, B220, FoxP3, or TCR alpha/beta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1 , Ly6G, Sca1, and TCR gamma/delta, and not C-kit, B220, FoxP3, or TCR alpha/beta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD49b, CD62L, NK1.1 , Ly6G, and TCR gamma/delta, and not C-kit, B220, FoxP3, or TCR alpha/beta. In some preferred embodiments, the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3, CD4, CD8, CD45, CD56, CD62L, NK1.1 , Ly6G, and TCR gamma/delta, and not C-kit, B220, FoxP3, or TCR alpha/beta.

In some particularly preferred embodiments, the population of NKT cells is characterized in that at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells express CD3, CD45, and/or CD56. can do. In some such embodiments, at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the NKT cells are CD3+/bright or CD3+/very bright, and/or CD45+/dark, and/or CD56+ to be.

In some embodiments, T cells of the present disclosure are capable of expressing CD3, CD4, CD45, and/or CD49b (human CD56). In some embodiments, T cells of the present disclosure are capable of expressing CD3, CD4, CD45, CD49b (human CD56), and/or TCR gamma/delta. In some embodiments, T cells of the disclosure are capable of expressing CD3, CD4, CD45, CD49b (human CD56), and/or TCR alpha/beta. In some embodiments, T cells of the present disclosure are capable of expressing CD3, CD4, CD45, CD49b (human CD56), and TCR gamma/delta. In some embodiments, T cells of the present disclosure are capable of expressing CD3, CD4, CD45, CD49b (human CD56), and TCR alpha/beta. In some embodiments, T cells of the present disclosure may not express CD8.

In embodiments related to the population of T cells of the present disclosure, the population of T cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD3, CD4, CD45, and / or CD49b (human CD56). In some embodiments, the population of T cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD3, CD4, CD45, CD49b (human CD56), and/or It may be characterized by expressing TCR gamma/delta. In some embodiments, the population of T cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells are CD3, CD4, CD45, CD49b (human CD56), and/or It may be characterized by expressing TCR alpha/beta. In some such embodiments, the population of T cells can be characterized as at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the T cells do not express CD8.

In some embodiments, the expression pattern of the surface protein can be determined by flow cytometry at 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours after administration of a glucocorticoid receptor (GR) modulator to the subject. In some embodiments, the expression pattern of a surface protein can be determined by flow cytometry (alone or in combination) using the equipment, reagents and/or conditions described herein.

Gamma delta T cells

Gamma delta T cell surface marker properties include CD3, CD4, CD8, CD69, CD56, CD27, CD40, CD40L, CD45RA, CD45, CD83, CD16, CD16a, CD16b, ICOS, CD161, Fas, CLEC7A/Dectin-1, FasL, Icadherin, IL-18R alpha, IL-23R, NKG2D/CD314, NKG2E, ocludin, TKR2, TRAIL, TCR-Vg9, TCR-Vd2, TCR-Vd1, TCR-Vd3, TCR-pan g/d, NKG2D, monoclonal chemokine receptor antibodies CCR5, CCR6, CCR7, CXCR3, CXCR4, or CXCR5 or combinations thereof. The surface marker properties of the cells of the invention may include one/or more of these. Gamma delta T cells are CCL2/JE/MCP-1, CXCL13/BLC/BCA-1, beta-defensin 2, beta-defensin 3, alpha-defensin 1, EGF, KGF/FGF-7, FGF-10. , GM-CSF, Granulisin, Granzyme A, Granzyme B, IFN-gamma, IGF-I/IGF-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL -12, IL-12/IL-23 p40, IL-12 p70, IL-13, IL-17/IL-17A, IL-22, IL-6/IL-6R alpha complex, LAP (TGF-beta 1) , TGF-beta, and/or TNF-alpha. The cells of the invention may secrete one/or more of these.

ICAM3 modulators in the context of the present disclosure are those that bind ICAM3 and promote the induction and/or recruitment of NKT cells, T cells, and dendritic cells of the invention. The ICAM3 modulator may be an ICAM3 antagonist/ICAM3 inhibitor, or may be an ICAM3 agonist/activator.

Such ICAM3 modulators may include, for example, anti-ICAM3 antibodies directed against ICAM3 or a portion thereof, small molecule modulators of ICAM3 (eg activators or inhibitors of ICAM3), and peptide agonists/proteins that bind ICAM3. have. Appropriate means of identifying ICAM3 modulators will be well known to those skilled in the art - for example, an anti-ICAM3 antibody may be prepared by contacting an ICAM3 epitope with a library of antibody molecules, and one or more specific antibodies of the library capable of binding to said epitope. may be identified by a method that may include selecting the molecule. Alternatively, they can be identified using, for example, competition binding assays using known anti-ICAM3 antibodies with competition determined using ELISA or flow cytometry. Similarly, small molecule modulators of ICAM3 can be identified by routine screening experiments such as radioligand binding assays and functional assays.

As already described above, the authors discovered the surprising ability of glucocorticoid receptor modulators (eg dexamethasone and other glucocorticoids) to bind to and exert a modulatory action on ICAM3. Thus, in some embodiments, the ICAM3 modulator may be a glucocorticoid receptor (GR) modulator. In some embodiments, the ICAM3 modulator may be a glucocorticoid, such as dexamethasone or betamethasone.

As used herein, the term glucocorticoid receptor (GR) modulator includes glucocorticoids, glucocorticoid receptor agonists, and any compound that binds to the glucocorticoid receptor. Glucocorticoid receptor (GR) modulators, such as glucocorticoids, exert their effects through membrane GR and cytoplasmic GR, which activate or inhibit gene expression. Some of the desirable lymphatic depleting effects of glucocorticoids and GR modulators are believed to be mediated through membrane GR or other non-genomic effects in addition to genomic effects. Glucocorticoids have been reported to have various effects on lymphocyte levels depending on the administered glucocorticoid concentration and treatment duration. In general, at low doses commonly used for chronic treatment, glucocorticoids have been reported to redistribute lymphocytes from the peripheral blood to the bone marrow, and at intermediate doses, glucocorticoids are reported to redistribute lymphocytes from the bone marrow, spleen and thymus into the peripheral blood and leukocyte redistribution. It has been reported to induce leukocytosis, which is believed to be The duration of effect also depends on the dosage level; For example, Fauci et al ( 1976) report that a single oral dose of 0.24 mg/kg dexamethasone suppressed peripheral blood T and B lymphocytes by 80%, recovery started at 12 hours and normal levels after 24 hours. We previously reported (in International Patent Application PCT/US2019/054395) that an acute oral dose of 3 mg/kg or more of dexamethasone was required to reduce peripheral blood T and B cells 24-48 hours after administration, and approximately 5 after administration. A return to baseline levels occurring on days −14 was demonstrated.

Glucocorticoid receptor (GR) modulators that may be used in the disclosed methods include, for example, selective glucocorticoid receptor modulators (SEGRM) and selective glucocorticoid receptor agonists (SEGRA). Glucocorticoids, selective glucocorticoid receptor modulators, and selective glucocorticoid receptor agonists (SEGRAs) that can be used in the disclosed methods are well known to those of skill in the art.

Some such glucocorticoids include, but are not limited to, dexamethasone, dexamethasone containing agents, hydrocortisone, methylpredisone, prednisone, corticone, budesonide, betamethasone, and beclomethasone. Other glucocorticoids include prednisolone, mometasone furoate, triamcinolone acetonide, and methylprednisolone.

Thus, in some embodiments of the methods of the present disclosure, the glucocorticoid receptor (GR) modulator may be a glucocorticoid. In some such embodiments, the glucocorticoid may be selected from the group consisting of: dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide, betamethasone, flumethasone and beclomethasone. In some preferred embodiments, the glucocorticoid may be selected from the group consisting of: dexamethasone, betamethasone, and methylprednisone. In some particularly preferred embodiments the glucocorticoid may be dexamethasone or betamethasone.

In some embodiments of the methods of the present disclosure, the glucocorticoid may be selected from the group consisting of: dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicoti. Nate, dexamethasone-21-acetate, dexamethasone phosphate, dexamethasone-21-phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate, dexamethasone palmitate, dexamethasone-21-palmitate Ropionate, dexamethasone propionate, dexamethasone acetate anhydrous, dexamethasone-21-phenylpropionate, dexamethasone-21-sulfobenzoate, dexamethasone hemo-sulfate, dexamethasone sulfate, dexamethasone beloxyl, dexamethasone acefurate, dexamethasone acefurate, Dexamethasone carboxyimide, dexamethasone cifesylate, dexamethasone 21-phosphate disodium salt, dexamethasone mesylate, dexamethasone linoleate, dexamethasone glucoside, dexamethasone glucuronide, dexamethasone iodine acetate, dexamethasone oxetanone, carboxymethylthio-dexamethasone, Dexamethasone bisethoxime, dexamethasone epoxide, dexamethasone linoleidate, dexamethasone methylorthovalerate, dexamethasone spermine, 6-hydroxy dexamethasone, dexamethasone tributylacetate, dexamethasone aspartic acid, dexamethasone galactopyranose, dexamethasone hydrochloride , hydroxy dexamethasone, carboxy dexamethasone, desoxy dexamethasone, dexamethasone butazone, dexamethasone cyclodextrin, dihydro dexamethasone, oxodexamethasone, propionyloxy dexamethasone, dexamethasone galactodi, dexamethasone alltamethasone, dexamethasone sodium dexamethasone al- , dexamethasone fibrate, dexamethasone tridecylate, dexamethasone crotonate, dexamethasone methanesulfonate, dexamethasone butyl acetate, dehydrodexamethasone, dexamethasone isothiocyanatoethyl)thioether, dexamethasone bromoacetate, dexamethasone he Any combination treatment, including the forms of miglutarate, deoxydexamethasone, dexamethasone chlorambusylate, dexamethasone melpalanate, formyloxy dexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and dexamethasone. In some preferred embodiments, the glucocorticoid may be dexamethasone base or dexamethasone sodium phosphate.

In some embodiments of the present disclosure, the glucocorticoid receptor modulator may not be one or more of the aforementioned agents.

In the methods of the present disclosure, the glucocorticoid receptor (GR) modulator or ICAM3 modulator is administered at a dose equivalent to approximately at least 6 mg/kg human equivalent dose (HED) of dexamethasone base.

Equivalent doses of other glucocorticoids or glucocorticoid receptor modulators can be calculated quickly and easily using publicly available corticoid transformation algorithms, preferably http://www.medcalc.com. As an example, 3 to 12 mg/kg dexamethasone is converted to 19 to 75 mg/kg prednisone. Prednisone has a biological half-life of about 20 hours, whereas dexamethasone has a biological half-life of about 36-54 hours, so prednisone would be administered at an equivalent biological dose of 19-75 mg/kg every 24 hours. More specifically, dexamethasone at a dose of 12 mg/kg corresponds to prednisolone at a dose of 75 mg/kg, which should be repeated about 2 to about 3 times every 24 hours. Betamethasone at a dose of 10 mg/kg is about 12 mg/kg dexamethasone and has a pharmacodynamic (biological) half-life similar to that of dexamethasone.

In the examples of this application, dexamethasone doses are given as human equivalent doses (HED). Methods for calculating human equivalent dose (HED) are known in the art. For example, the FDA's Center for Drug Evaluation and Research (CDER) published a highly cited guidance document (U.S. Department of Health CDER, 2005) in 2005, which lists animal doses based on body surface area in Table 1 on page 7 of that document. We present an established algorithm (a generally accepted method for extrapolating interspecies dose) for conversion to HED. For reference, Table 1 below is reproduced. One of ordinary skill in the art understands that the animal dose, HED, in mg/kg, described below is easily calculated using the standard conversion factors in the right column of Table 1:

Table 1: Conversion of animal doses to human equivalent doses based on body surface area

In some embodiments of the methods of the present disclosure, the glucocorticoid receptor (GR) modulator or ICAM3 modulator is administered at a dose equivalent to approximately at least 12 mg/kg human equivalent dose (HED) of dexamethasone base. In another preferred embodiment, the glucocorticoid receptor (GR) modulator is administered at a dose equivalent to about at least 15 mg/kg or about at least 18 mg/kg human equivalent dose (HED) of dexamethasone base. In another preferred embodiment, the glucocorticoid receptor (GR) modulator is administered at a dose equivalent to approximately at least 21 mg/kg or at least about 24 mg/kg human equivalent dose (HED) of dexamethasone base. In some preferred embodiments, the glucocorticoid receptor (GR) modulator is about 12 mg/kg human equivalent dose (HED) of dexamethasone base, about 15 mg/kg human equivalent dose (HED) of dexamethasone base, or about 18 dexamethasone base mg/kg human equivalent dose (HED), or about 21 mg/kg human equivalent dose of dexamethasone base (HED) or about 24 mg/kg human equivalent dose of dexamethasone base (HED), or about 30 mg/kg of dexamethasone base It is administered at a human equivalent dose (HED), or at a dose equivalent to about 45 mg/kg human equivalent dose (HED) of dexamethasone base.

In some embodiments of the methods of the present disclosure, the glucocorticoid receptor (GR) modulator or ICAM3 modulator is administered at an approximately at least 6-45 mg/kg human equivalent dose (HED) of dexamethasone base; approximately at least 15-24 mg/kg human equivalent dose (HED) of dexamethasone base; approximately at least 6-12 mg/kg human equivalent dose (HED) of dexamethasone base; or approximately at least 12-15 mg/kg human equivalent dose (HED) of dexamethasone base; or approximately at least 18-30 mg/kg human equivalent dose (HED) of dexamethasone base; or at a dose equivalent to approximately at least 15-18 mg/kg human equivalent dose (HED) of dexamethasone base. In embodiments wherein the infectious disease is a disease caused by a coronavirus, eg, COVID-19 infection, the glucocorticoid receptor (GR) modulator is preferably equivalent to about 18-30 mg/kg human equivalent dose (HED) of dexamethasone base. It may be administered in a dose.

In the methods of the present disclosure, the glucocorticoid receptor (GR) modulator or ICAM3 modulator may be administered in a single acute dose, or in a total dose given over about 24, 48, or 72 hours. In some preferred embodiments, the glucocorticoid receptor (GR) modulator is administered in a single acute dose. In another preferred embodiment, the glucocorticoid receptor (GR) modulator is administered in a total dose provided over a period of about 72 hours.

In some embodiments having, suspected of having, or having been diagnosed with an infectious disease, such as a disease caused by a coronavirus (eg, COVID-19) infection, a glucocorticoid receptor modulator (preferably dexamethasone or betamethasone) can be administered as a solution in an aqueous medium. In some such embodiments, the glucocorticoid receptor modulator is provided at a concentration equal to about 24 mg/ml dexamethasone phosphate (20 mg/ml dexamethasone base; 26.2 mg/ml dexamethasone sodium phosphate), and about 18-30 mg/ml of dexamethasone base It may be administered by intravenous (IV) infusion over a period of about 1-2 hours with a maximum target dose of kg human equivalent dose (HED). In another embodiment, the glucocorticoid receptor modulator is provided as dexamethasone tablets dissolved in orange juice or citric acid (pH 3.3-4.2) and orally at a maximum target dose of about 18-30 mg/kg human equivalent dose (HED) of dexamethasone base. or via a gavage.

In some embodiments of the methods of the present disclosure, a method of generating a population of natural killer T cells (NKT cells), a method of generating a population of T cells, and/or a method of generating or activating a population of dendritic cells are provided in a subject. further administering to the patient one or more additional doses of a glucocorticoid receptor (GR) modulator or an ICAM3 modulator.

In this context, one or more doses are administered in addition to the first or previous dose of the glucocorticoid receptor (GR) modulator or ICAM3 modulator and thus may be termed subsequent or second, third, fourth doses, etc. Thus, in some embodiments, one or more additional doses may be administered about 24, 48, 72, 96, 120, 144, or 168 hours after the previous dose (administration). In some embodiments, one or more additional doses may be administered about every 24, 48, 72, 96, 120, 144, or 168 hours after the previous dose (administration). In some other embodiments, the one or more additional doses may be administered once a week, once every two weeks, once every three weeks, or once a month after the previous dose (administration). In some other embodiments, one or more additional doses may be administered twice weekly after the previous dose (administration).

In some embodiments, one or more additional doses may be administered between about 24 hours and 168 hours after the previous dose (administration). In other embodiments, the one or more additional doses may be administered between about 24 hours and 120 hours, between about 24 hours and 72 hours, or between about 24 hours and 48 hours after the previous dose (administration). In some other embodiments, the one or more additional doses may be administered between about 48 hours and 168 hours, between about 48 hours and 120 hours, or between about 48 hours and 72 hours after the previous dose (administration). In some other embodiments, the one or more additional doses may be administered between about 72 hours and 168 hours, or between about 72 hours and 120 hours after the previous dose (administration).

In some embodiments, the subsequent dose is given 7 days after the initial dose. In some embodiments, the subsequent dose is given 14 days after the initial dose. In some embodiments, the subsequent dose is given 21 days after the initial dose.

In some embodiments, the subject has, is suspected of having, or is diagnosed with T-cell lymphoma, one or more additional doses are administered every 21 days, or every 14 days, or every 5-7 days for a period as may be determined by the physician. can do.

In some embodiments, the subject has, is suspected of having, or is diagnosed with B-cell lymphoma, one or more additional doses are administered every 21 days, or every 14 days, or every 5-7 days for a period as may be determined by the physician. can do.

In some embodiments of the methods of the present disclosure, the method of producing a population of natural killer T cells (NKT cells) can further comprise administering to the subject a NKT cell activator. As used herein, the term NKT cell activator includes any agent or molecule that triggers the activation of NKT cells. Activation of NKT cells is associated with activation markers and upregulation of Th1 and Th2 cytokines and chemokines. NKT cell activators that can be used in the disclosed methods are well known to those of skill in the art.

Some of these NKT cell activators include adipokine, leptin, adiponectin, apelin, kemerin, MCP-1, PAI-1, RBP4, bispartin, omentin, vaspin, progranulin, CTRP-4, cytomegalovirus. Cain, IL-1α, IL-1β, IL-1RA. IL-18, IL-33, IL-36α, IL-36β, IL-36γ. IL-36RA, IL-37, IL-38, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IFN-α, IFN-β, IFN-δ, IFN- ε, IFN-κ, IFN-τ, IFN-ω, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, IL-6, IL-11, IL-31, CLCF1, CNTF, Leptin, LIF, OSM, iL-12, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, 4-1BBL, BAFF, CD40LG, CD70, CD95L/CD178, EDA- A1, LTA/TNF-β, TNF-α, TNFSF4, TNFS8, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TGF-β1, TGF-β2, TGF-β3, IL-13, G-CSF, GM-CSF, CSF1. Chemokine, CXCL1-CXCL17, CC, CCL1-CCL28, CX3CL1, XCL1, XCL2, myokine, BDNF, decorin, irisin, myostatin, myonectin, osteonectin, prostaglandin, PGI2, PGD2, PGE2, PGF2α, pro Stamide, Prostamide I2, Prostamide D2, Prostamide E2, Prostamide F2α, Virokine, Growth Factor, Adrenomedulin, Angiopoietin, Autocrine Motor Factor, Osteogenic Protein, Cilia Neurotrophic factor, leukemia inhibitory factor, M-CSF, EGF, Ephrin A1-A5, Ephrin B1-B3, Erythropoietin, FGF1-FGF23, Fetal bovine somatotropin, GDNF, Neuroturin, Persepin, Arte Min, growth differentiation factor-9, hepatocyte growth factor, hepatocyte-derived growth factor, insulin, insulin-like growth factor ½, keratinocyte growth factor, migration promoter, macrophage-stimulating protein, neuregulin 1-4, neurotrophin ¾ , nerve growth factor, placental growth factor, platelet-derived growth factor, elongation enzyme, T cell growth factor, TGF-α, TGF-β, VEGF, Wnt signaling pathway, anti-NKG2D antibody or its ligand MICA (MHC class I chain Related sequence A), DNAM-1 binding, 4-1BB binding, PD-1 inhibitor, NKT activator, α-galactosylceramide, α-glucuronosylceramide, α-galturoncilceramide, α-galactosyldia Silgiocerol, phosphatidylinositol-manosidase, α-glutosyldiacylglycerol, cholesterol α-glucoside, β-glucosylceramide, isoglovotrihexosylceramide, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, house dust extract, GSL-1, NKp44L, ULBP, pathogen-derived molecular structure, PAMP, LPS, pathogen-derived RNA, pathogen-derived DNA, viral ligand, synthetic α-galacosylceramide, KRN7000, PBS44, PBS57, anti-inflammatory agent, IL-10, IL -19, IL-20, IL-22, IL-24, IL-28A, IL-28B, IL-29 goes

In some embodiments of the present disclosure, the NKT cell activator may not be one or more of the agents recited above.

After activation, NKT cells express NKp46 (human NKp44), low CD3 and CD49b expression and IL-10, TGF-β, IFNgamma, IL-4 and several Th1 and Th2 cytokines, human class I restricted T cells Related Molecules (CRTAM), CCL3/MIP1a, CCL4/MIP1h and CCL5/Lantes and XCL1/Lymphotactin, Granzyme, CD45RO+ CD62L+, CD25, IL2Rbeta, GM-CSF, IL-2, IL-13, TNFalpha, It expresses IL-17, IL-21, CD44, CD69, and IL-22. Also, in the tumor environment, NKT cells are organized into lines migrating towards the tumor cells on all sides.

In some preferred embodiments of the methods of the present disclosure, the NKT cell activator is alpha GalCer (alpha-galactosylceramide; α-GalCer) sulfatide (3-O-sulfogalactosylceramide; SM4; galactosulfate) cerebroside), or NKT-activating antibody, or perforin, nitric oxide, IL-2, interferon alpha and gamma, TGFbeta, TNFalpha, TNFbeta, G-CSF, VEGF, FGF -18, IL-17, CXCL5, CXCR2, CXCR5, CCR4-CCL17/22, CCR8-CCL1, CCR10-CCL28, and CXCR3-CCL9/10/11, CCL5, CXCR9, CCL2, CCL3, CCL4, CCL5, CXCL9 or CXCL10, interferon (IFN) γ-inducible chemokines CXCL9, CXCL10, and CXCL11, CCL5 and CXCL9, CCR5, IL-32, IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF , MCP-1, MCP-3, IP-10, MIG, or MIP-1α. In some other preferred embodiments of the methods of the present disclosure, the NKT cell activator may be alpha GalCer loaded dendritic cells or monocytes. In some embodiments of the methods of the present disclosure, the NKT cell activator is within 1,3, 24, 48, 72, 96, 120, 144, or 168 hours after administration of a dose of a glucocorticoid receptor (GR) modulator or ICAM3 modulator. may be administered. In some preferred embodiments the NKT cell activator may be administered within 1, 3, or 48 hours or before or after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator dose. In some particularly preferred embodiments the NKT cell activator may be administered within 1, 3, or 48 hours after administration of the glucocorticoid dose or before or after.

In some embodiments of the methods of the present disclosure, the method of generating a population of T cells can further comprise administering to the subject a T cell activator. As used herein, the term T cell activator includes any agent or molecule that triggers the activation of T cells. T cells can be activated through the interaction of TCRs with antigenic peptides and MHCs and through non-antigen specific costimulatory agents (eg cytokine interleukin 1). Activation of T cells is associated with increased cytokine and chemokine production, induction of dendritic cell maturation, macrophage recruitment, and increased cytolytic activity. Activation of gamma delta T cells may also be associated with increased production of growth factors (eg IGF-1, VEGF and FGF-2) that maintain epidermal integrity, as well as antigen presentation to alpha beta T cells. Activation of T cells may also be associated with changes in the expression pattern of surface markers. For gamma delta T cells, this may comprise one or more of the following marker phenotypes: CD5-, CD4-/CD8- (double negative), CD3+, CD69, CD56, CD27, CD45RA+, CD45, TCR-Vg9+, TCR -Vd2+, TCR-Vd1+, and/or TCR-Vd3+. T cell activators that may be used in the disclosed methods are well known to those of skill in the art.

Some of these T cell activators are adipokine, leptin, adiponectin, apelin, kemerin, MCP-1, PAI-1, RBP4, bispartin, omentin, vaspin, progranulin, CTRP-4, cytotoxic. Cain, IL-1α, IL-1β, IL-1RA. IL-18, IL-33, IL-36α, IL-36β, IL-36γ. IL-36RA, IL-37, IL-38, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IFN-α, IFN-β, IFN-δ, IFN- ε, IFN-κ, IFN-τ, IFN-ω, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, IL-6, IL-11, IL-31, CLCF1, CNTF, Leptin, LIF, OSM, iL-12, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, 4-1BBL, BAFF, CD40LG, CD70, CD95L/CD178, EDA- A1, LTA/TNF-β, TNF-α, TNFSF4, TNFS8, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TGF-β1, TGF-β2, TGF-β3, IL-13, G-CSF, GM-CSF, CSF1. Chemokine, CXCL1-CXCL17, CC, CCL1-CCL28, CX3CL1, XCL1, XCL2, myokine, BDNF, decorin, irisin, myostatin, myonectin, osteonectin, prostaglandin, PGI2, PGD2, PGE2, PGF2α, pro Stamide, Prostamide I2, Prostamide D2, Prostamide E2, Prostamide F2α, Virokine, Growth Factor, Adrenomedulin, Angiopoietin, Autocrine Motor Factor, Osteogenic Protein, Cilia Neurotrophic factor, leukemia inhibitory factor, M-CSF, EGF, Ephrin A1-A5, Ephrin B1-B3, Erythropoietin, FGF1-FGF23, Fetal bovine somatotropin, GDNF, Neuroturin, Persepin, Arte Min, growth differentiation factor-9, hepatocyte growth factor, hepatocyte-derived growth factor, insulin, insulin-like growth factor ½, keratinocyte growth factor, migration promoter, macrophage-stimulating protein, neuregulin 1-4, neurotrophin ¾ , nerve growth factor, placental growth factor, platelet-derived growth factor, elongation enzyme, T cell growth factor, TGF-α, TGF-β, VEGF, Wnt signaling pathway, NKT activator, α-galactosylceramide, α- Glucoronosylceramide, α-galturoncilceramide, α-galactosyldiacyl grouprol, phosphatidylinositol-manosidase, α-glutosyldiacylglycerol, cholesterol α-glucoside, β-glucosylceramide, iso Globotrihexosylceramide, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, house dust extract, GSL-1, NKp44L, ULBP, pathogen-derived molecular structure, PAMP, LPS, pathogen-derived RNA, pathogen-derived DNA, viral ligand, synthetic α- Galcosylceramide, KRN7000, PBS44, PBS57, Anti-inflammatory, IL-10, IL-19, IL-20, IL-22, IL-24, IL-28A, IL-28B, IL-29 does not

In some embodiments of the present disclosure, the T cell activator may not be one or more of the agents recited above.

In some embodiments of the methods of the present disclosure, the T cell activator is administered within 1, 3, 24, 48, 72, 96, 120, 144, or 168 hours after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator dose. can be In some preferred embodiments the T cell activator may be administered within or before or after 1, 3, or 48 hours after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator dose. In some particularly preferred embodiments the T cell activator may be administered within or before or after 1, 3, or 48 hours after administration of the glucocorticoid dose.

In some embodiments of the methods of the present disclosure, the method of activating a population of dendritic cells can further comprise administering to the subject a dendritic cell activator. As used herein, the term dendritic cell activator includes any agent or molecule that triggers dendritic activation. Dendritic cells can be activated directly by conserved pathogen molecules and indirectly by inflammatory mediators (eg, those produced by other cell types that recognize such molecules). Activation of dendritic cells is associated with loss of adhesive structures, reorganization of the cytoskeleton, and increased cell motility. Activation is also associated with a decrease in endocytic activity but with increased expression of MHC-II and co-stimulatory molecules required for T cell activation. Activation of dendritic cells may also be associated with changes in the expression pattern of surface markers. For CD11b+ dendritic cells, this may comprise one or more of the following marker phenotypes: CD4-, CD8-, CD11c+, CLEC9a-, CX3CR1+, EpCAM/TROP1-, F4/80+, Fcg RI/CD64+, integrin aE/ CD103-, integrin aM/CD11b+, Langerin/CD207-, MHC class II+, SIRPa/CD172a+, XCR1. Dendritic cell activators that can be used in the disclosed methods are well known to those skilled in the art.

Some such dendritic cell activators include adipokine, leptin, adiponectin, apelin, kemerin, MCP-1, PAI-1, RBP4, bispartin, omentin, vaspin, progranulin, CTRP-4, cytomegalovirus. Cain, IL-1α, IL-1β, IL-1RA. IL-18, IL-33, IL-36α, IL-36β, IL-36γ. IL-36RA, IL-37, IL-38, IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, IFN-α, IFN-β, IFN-δ, IFN- ε, IFN-κ, IFN-τ, IFN-ω, IFN-γ, IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4, IL-6, IL-11, IL-31, CLCF1, CNTF, Leptin, LIF, OSM, iL-12, IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, IL-17F, 4-1BBL, BAFF, CD40LG, CD70, CD95L/CD178, EDA- A1, LTA/TNF-β, TNF-α, TNFSF4, TNFS8, TNFSF10, TNFSF11, TNFSF12, TNFSF13, TNFSF15, TGF-β1, TGF-β2, TGF-β3, IL-13, G-CSF, GM-CSF, CSF1. Chemokine, CXCL1-CXCL17, CC, CCL1-CCL28, CX3CL1, XCL1, XCL2, myokine, BDNF, decorin, irisin, myostatin, myonectin, osteonectin, prostaglandin, PGI2, PGD2, PGE2, PGF2α, pro Stamide, Prostamide I2, Prostamide D2, Prostamide E2, Prostamide F2α, Virokine, Growth Factor, Adrenomedulin, Angiopoietin, Autocrine Motor Factor, Osteogenic Protein, Cilia Neurotrophic factor, leukemia inhibitory factor, M-CSF, EGF, Ephrin A1-A5, Ephrin B1-B3, Erythropoietin, FGF1-FGF23, Fetal bovine somatotropin, GDNF, Neuroturin, Persepin, Arte Min, growth differentiation factor-9, hepatocyte growth factor, hepatocyte-derived growth factor, insulin, insulin-like growth factor ½, keratinocyte growth factor, migration promoter, macrophage-stimulating protein, neuregulin 1-4, neurotrophin ¾ , nerve growth factor, placental growth factor, platelet-derived growth factor, elongation enzyme, T cell growth factor, TGF-α, TGF-β, VEGF, Wnt signaling pathway, NKT activator, α-galactosylceramide, α- Glucoronosylceramide, α-galturoncilceramide, α-galactosyldiacyl grouprol, phosphatidylinositol-manosidase, α-glutosyldiacylglycerol, cholesterol α-glucoside, β-glucosylceramide, iso Globotrihexosylceramide, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, house dust extract, GSL-1, NKp44L, ULBP, pathogen-derived molecular structure, PAMP, LPS, pathogen-derived RNA, pathogen-derived DNA, viral ligand, synthetic α- Galcosylceramide, KRN7000, PBS44, PBS57, Anti-inflammatory, IL-10, IL-19, IL-20, IL-22, IL-24, IL-28A, IL-28B, IL-29 does not

In some embodiments of the present disclosure, the dendritic cell activator may not be one or more of the agents recited above.

In some embodiments of the methods of the present disclosure, the dendritic cell activator may be administered within 24, 48, 72, 96, 120, 144, or 168 hours after administration of a dose of a glucocorticoid receptor (GR) modulator or ICAM3 modulator. . In some preferred embodiments the dendritic cell activator may be administered within 48 hours or before or after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator dose. In some particularly preferred embodiments the dendritic cell activator may be administered within 48 hours or before or after administration of the glucocorticoid dose.

The terms “subject” and “patient” are used interchangeably herein and refer to a human or animal. In some embodiments of the methods of the present disclosure, the subject can be a mammal. In some preferred embodiments, the subject may be a human of any gender or race. In some embodiments, the human is an adult human. In some embodiments of the methods of the present disclosure, the subject may be a healthy subject, such as a healthy adult human subject. A healthy subject in this context is a subject that does not have the disease.

In some embodiments of the methods of the present disclosure, the subject may have, suspected of having, or been diagnosed with a disease selected from the group consisting of cancer, an autoimmune disease, or an infectious disease (also referred to as a microbial disease).

As used herein, “cancer” refers to a disease characterized by the uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body either locally or through the bloodstream and lymphatic system. Examples of various cancers are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and the like. The terms “tumor” and “cancer” are used interchangeably herein, eg, both terms include solid and liquid, eg, diffuse or circulating tumors. As used herein, the term “cancer” or “tumor” includes premalignant as well as malignant cancers and tumors.

In some embodiments of the present disclosure, the cancer may be: malignant neoplasm of the lips, malignant neoplasm of the esophagus, malignant neoplasm of the tongue, malignant neoplasm of the gums, malignant neoplasm of the oral cavity, malignancy of the parotid gland Neoplasm, salivary gland malignant neoplasm, pharyngeal malignant neoplasm, esophageal malignant neoplasm, malignant neoplasm, small intestine malignant neoplasm, colon malignant neoplasm, rectal sigmoid colon malignancy, rectal malignant neoplasm, anal malignant neoplasm Organism, hepatic malignancy, gallbladder malignant neoplasm, biliary tract malignant neoplasm, pancreatic malignant neoplasm, intestinal malignant neoplasm, spleen malignant neoplasm, nasal and middle ear malignant neoplasm, sinus malignant neoplasm , malignant neoplasms of the larynx, malignant neoplasms of organs, malignant neoplasms of the bronchi and lungs, malignant neoplasms of the thymus, malignant neoplasms of the heart, mediastinum and pleura, malignant neoplasms of the respiratory and thoracic organs, bone and extremities Malignant neoplasms of articular cartilage, malignant neoplasms of skull and facial bones, malignant neoplasms of spine, malignant neoplasms of ribs, sternum and clavicle, malignant neoplasms of pelvic bones, sacrum and coccyx, malignant melanoma of skin, lips malignant melanoma of the, malignant melanoma of the eyelids, including canthus, malignant melanoma of the ear and external auditory meatus, malignant melanoma of the face, malignant melanoma of the skin of the anus, malignant melanoma of the skin of the breast, malignancy of the limbs including the shoulder Melanoma, Merkel cell carcinoma, basal cell carcinoma of the skin of the lips, squamous cell carcinoma of the skin of the lips, eyelid other and unspecified malignancies including skin/canthus, skin/ear and external auditory canal malignancies, skin/and details Malignant neoplasms of other and unspecified areas of the face, other and unspecified, basal cell carcinoma of the skin of other and unspecified areas of the face, squamous cell carcinoma of the skin of the face and unspecified areas of the face, basal cell carcinoma of the skin of the scalp and neck, of the skin of the scalp and neck squamous cell carcinoma, basal cell carcinoma of the skin of the trunk, basal cell carcinoma of the skin of the anus, basal cell carcinoma of the skin of the breast, squamous cell carcinoma of the skin of the trunk, squamous cell carcinoma of the skin of the anus, squamous cell carcinoma of the skin of the breast, other parts of the trunk squamous cell carcinoma of the skin extremity including skin/shoulder Other and unspecified malignant neoplasms, extremity basal cell carcinoma of the extremities including skin/shoulder, including skin/shoulder Squamous cell carcinoma of the extremities, basal cell carcinoma of the skin of the extremities including the buttocks, squamous cell carcinomas of the skin of the extremities including the buttocks, mesothelioma, Kaposi's sarcoma, malignant neoplasms of the peripheral and autonomic nervous system, malignant neoplasms of the retroperitoneum and peritoneum , other malignant neoplasms of connective and soft tissues, malignant neoplasms of thoracic connective and soft tissues, malignant neoplasms of connective and soft tissues of the abdomen, malignant neoplasms of connective and soft tissues of the pelvis, malignant neoplasms of trunk and soft tissues, unspecified; Malignant neoplasm of overlap of connective and soft tissue, unspecified connective and soft tissue malignant neoplasm, gastrointestinal stromal tumor, malignant neoplasm of breast, malignant neoplasm of vulva, malignant neoplasm of vagina, malignant neoplasm of cervix, Malignant neoplasm of the uterine body, malignant neoplasm of the uterus, unspecified, malignant neoplasm of the ovary, other and unspecified malignant neoplasm of the female genital tract, malignant neoplasm of the placenta, malignant neoplasm of the penis, malignant neoplasm of the prostate malignant neoplasm of testis, other and unspecified male genital malignancy, renal malignant neoplasm, renal pelvis malignant neoplasm, ureter malignant neoplasm, bladder malignant neoplasm, other and unspecified urinary tract malignancy Malignant neoplasm of organs, malignant neoplasm of eye and appendages, malignant neoplasm of meninges, malignant neoplasm of brain, malignant neoplasm of spinal cord, cranial nerve, malignant neoplasm of optic nerve, malignant neoplasm of cranial nerve other and unspecified, Unspecified central nervous system malignant neoplasm, thyroid malignant neoplasm, adrenal gland malignant neoplasm, endocrine gland and related structures malignant neoplasm, malignant neuroendocrine tumor, malignant carcinoid tumor, secondary neuroendocrine tumor, head, face and neck Malignant neoplasms, malignant neoplasms of the chest, malignant neoplasms of the abdomen, malignant neoplasms of the pelvis, malignant neoplasms of the extremities, malignant neoplasms of the lower extremities, secondary and unspecified malignant neoplasms of lymph nodes, secondary to respiratory and gastrointestinal tract Malignant neoplasm, secondary malignant neoplasm of kidney and renal pelvis, secondary malignant neoplasm of bladder and other unspecified urinary tract, secondary malignant neoplasm of skin, secondary malignant neoplasm of brain and meninges, nervous system and unspecified part Secondary malignant neoplasm, secondary malignant neoplasm of bone and bone marrow, secondary malignant neoplasm of ovary, secondary malignant neoplasm of adrenal gland, Hodgkin's lymphoma, follicular lymphoma, non -follicular lymphoma, small cell B-cell lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, lymphoblastic (diffuse) lymphoma, Burkitt's lymphoma, other non-follicular lymphoma, non-follicular (diffuse) lymphoma, unspecified , mature T/NK-cell lymphoma, Sézary disease, peripheral T-cell lymphoma, unclassified, anaplastic large-cell lymphoma, ALK-positive, anaplastic large-cell lymphoma, ALK-negative, cutaneous T-cell lymphoma, unspecified, Other mature T/NK-cell lymphoma, unspecified, mature T/NK-cell lymphoma, other and unspecified types of non-Hodgkin's lymphoma, malignant immunoproliferative disease and certain other B-cell lymphoma, multiple myeloma and malignant plasma cells Neoplasia, lymphocytic leukemia, acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia of B cell type Prolymphocytic leukemia of B cell type, Hairy cell leukemia, adult T-cell lymphoma/leukemia (HTLV-1- related), prolymphocytic leukemia of T cell type, mature B cell leukemia Burkitt type, other lymphocytic leukemia, lymphocytic leukemia, unspecified, myeloid leukemia, acute myeloblastic leukemia, chronic myelogenous leukemia, BCR/ABL-positive , atypical chronic myeloid leukemia, BCR/ABL-negative, myeloid sarcoma, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute myeloid leukemia with 11q23- or higher, other myeloid leukemia, myeloid leukemia, unspecified myeloid leukemia, monocytic leukemia, Chronic myelomonocytic leukemia, childhood myelomonocytic leukemia, other monocytic leukemias, unspecified monocytic leukemia, other specific cell types leukemia, acute erythroblastic leukemia, acute megakaryocyte leukemia, mast cell leukemia, acute with myelofibrosis Panmyelopathy, myelodysplastic disease, unspecified, specific leukemia, unspecified cellular leukemia, unspecified cellular chronic leukemia, unspecified leukemia Other and unspecified lymphoma, hematopoietic tissue malignancy, oral cavity, esophagus and carcinoma in situ of the stomach, sigmoid colorectal carcinoma in situ, rectal carcinoma in situ, carcinoma in situ of the anus and anal canal, and carcinoma in situ of other and unspecified parts of the intestine , Carcinoma in situ of unspecified part of intestine, Carcinoma in situ of other intestine, Carcinoma in situ of liver, gallbladder and bile duct, Carcinoma in situ of certain other digestive organs, Carcinoma in situ of digestive tract, unspecified, Carcinoma in situ of middle ear and respiratory system, of the larynx Carcinoma in situ, carcinoma in situ of trachea, carcinoma in situ of bronchi and lungs, carcinoma in situ of other parts of the respiratory system, melanoma in situ, melanoma in situ, melanoma in situ, melanoma in situ of the eyelids including canthus, melanoma in situ of the ear and ear canal , Partial melanoma in situ of face, unspecified, melanoma in situ of scalp and neck, melanoma in situ of torso, melanoma in situ of anal skin, melanoma in situ of breast (skin) (soft tissue), upper extremity in situ including shoulder melanoma, melanoma in situ, including the buttocks, melanoma in situ, carcinoma in situ, carcinoma in situ of the skin of the lips, carcinoma in situ of the skin of the eyelids including canthus, carcinoma in situ of the ears and external auditory meatus, other and Carcinoma in situ of the skin of unspecified site, Carcinoma in situ of the skin of the scalp and neck, Carcinoma in situ of the skin of the trunk, Carcinoma in situ of the skin of the upper extremities including the shoulder, Carcinoma in situ of the skin of the lower extremities including the hip, Carcinoma in situ of the skin of the breast Carcinoma, lobular carcinoma in situ of breast, carcinoma in situ of breast, other types of breast carcinoma in situ, unspecified types, carcinoma in situ of breast, carcinoma in situ, carcinoma in situ of cervix, cervix in situ, cervix, unspecified Carcinoma in situ, other and unspecified genital carcinoma in situ, endometrial carcinoma in situ, vulvar carcinoma in situ, vaginal carcinoma in situ, endometrial cancer Other and unspecified carcinoma in situ of female genital tract, carcinoma in situ, carcinoma in situ of prostate, detailed Carcinoma in situ of male genitalia, unknown, carcinoma in situ of scrotum, other carcinoma in situ of male genitalia, carcinoma in situ of bladder, other and unspecified carcinoma in situ of urinary tract, carcinoma in situ of eye, carcinoma in situ of thyroid and other endocrine glands, oral cavity and benign neoplasms of the pharynx, benign neoplasms of the major salivary glands, benign neoplasms of the colon, rectum, anus and anal canals, benign neoplasms of the digestive system and ambiguity; Benign neoplasm of esophagus, benign neoplasm of stomach, benign neoplasm of duodenum, benign neoplasm of other and unspecified parts of small intestine, benign neoplasm of liver, benign neoplasm of extrahepatic bile duct, benign neoplasm of pancreas, benign neoplasm of endocrine pancreas Benign neoplasm, benign neoplasm of unknown site in digestive system, benign neoplasm of middle ear and respiratory system, benign neoplasm of respiratory system, unspecified benign neoplasm of other and unspecified internal thoracic organs, benign neoplasm of thymus Organisms, benign neoplasms of the heart, benign neoplasms of the mediastinum, benign neoplasms of certain other internal thoracic organs, benign neoplasms of unspecified internal thoracic organs, benign neoplasms of bone and articular cartilage, benign neoplasms of single bones of the upper extremities , benign neoplasm of long bone of lower extremity, benign neoplasm of single bone of lower extremity, benign neoplasm of skull and facial bones, benign neoplasm of lower jaw bone, benign neoplasm of spine, benign neoplasm of ribs, sternum and clavicle, pelvic bone, Benign neoplasm of sacrum and coccyx, benign neoplasm of bone and articular cartilage, unspecified, benign lipomatous neoplasm, subcutaneous Benn's lipomatous neoplasm of skin, head, face and neck, benign lipomatous neoplasm of thoracic organs , benign lipomatous neoplasm of internal organs, benign lipomatous neoplasm of spermatic cord, benign lipomatous neoplasm of other sites, benign lipomatous neoplasm of kidney, benign lipomatous neoplasm of other genitourinary organs, hemangioma and lymphangioma , all sites, hemangioma, hemangioma unspecified, hemangioma of skin and subcutaneous tissue, hemangioma of intracranial structure, hemangioma of intra-abdominal structure, hemangioma of other sites, lymphangioma, all sites, benign neoplasm of mesothelial tissue, retroperitoneum and Benign neoplasms of peritoneal soft tissue, other benign neoplasms of connective and other soft tissues, melanocyte nevus, melanocytic nevus of the lips, melanocytic nevus of the eyelid including canthus, melanocyte nevus of unspecified eyelids including canthus, ear and external auditory meatus melanocyte nevus, melanocyte nevus of other and unspecified parts of face, melanocytic nevus of scalp and neck, melanocytic nevus of trunk, melanocytic nevus of upper extremities including shoulder, melanocytic nevus of lower extremities including buttocks, unspecified Melanocyte nevus of the eye, including the canthus Other benign neoplasms of the eyelid skin, other benign neoplasms of the skin/ear and external auditory meatus, skin/other benign neoplasms of the left ear and external auditory meatus, other benign neoplasms of the skin other and unspecified areas of the face Other benign neoplasms of the skin other than the face Neoplasm, other benign neoplasms of the skin of the scalp and neck, other benign neoplasms of the skin of the trunk, other benign neoplasms of the upper extremities including skin/shoulder, other benign neoplasms of the skin of the lower extremities including the buttocks, other benign neoplasms of the skin, unspecified Neoplasia, benign neoplasm of breast, benign neoplasm of unspecified breast, leiomyoma of uterus, other benign neoplasm of uterus, benign neoplasm of ovary Other and unspecified benign neoplasm of female genitalia, benign male genitalia Neoplasm, benign neoplasm of the urinary tract, benign neoplasm of the kidney, benign neoplasm of the renal pelvis, benign neoplasm of the ureter, benign neoplasm of the bladder, benign neoplasm of the urethra, other benign neoplasms of the specific urinary tract, unspecified benign neoplasm of the urinary tract of Benign neoplasm, benign neoplasm of unspecified site of orbit, benign neoplasm of unspecified site of eye, benign neoplasm of meninges, benign neoplasm of brain and central nervous system, benign neoplasm of thyroid gland, other and unspecified Benign neoplasm of endocrine glands, benign neoplasms of other and unspecified sites, benign neoplasms of lymph nodes, benign neoplasms of peripheral nerves and autonomic nervous system, benign neoplasms of other specific sites, benign neuroendocrine tumors, other neuroendocrine tumors , neoplasm of unknown behavior of oral and digestive organs, neoplasm of unknown behavior of major salivary glands, neoplasm of unknown behavior of pharynx, neoplasm of unknown behavior of oral region, neoplasm of unknown behavior of stomach , neoplasm of unknown behavior of small intestine, neoplasm of unknown behavior of appendix, neoplasm of unknown behavior of colon, neoplasm of unknown behavior of rectum, neoplasm of unknown behavior of liver, GB & bile duct, Neoplasm of unknown behavior of other digestive organs, neoplasm of unknown behavior of digestive system, neoplasm of middle ear and thoracic organs, neoplasm of unknown behavior of larynx, trachea, Neoplasm of unknown behavior of bronchus and lungs, neoplasm of unknown behavior of pleura, neoplasm of unknown behavior of mediastinum, neoplasm of unknown behavior of thymus, neoplasm of unknown behavior of other respiratory organs, details Neoplasm of unknown respiratory behavior, unknown neoplasm of female genitalia, neoplasm of unknown behavior of uterus, neoplasm of ovary of unknown behavior, neoplasm of unknown ovarian behavior, Neoplasm of unknown placental behavior, male genital neoplasm of unknown behavior, urinary tract neoplasm of unknown behavior, renal neoplasm of unknown behavior, renal neoplasm of unknown behavior of unspecified Neoplasm of unknown behavior of ureter, neoplasm of unknown behavior of ureter, neoplasm of unknown behavior of bladder, other neoplasm of unknown behavior of urinary tract, Neoplasm of unknown behavior of the meninges, neoplasm of unknown behavior of the cerebral meninges, neoplasm of unknown behavior of the spinal meninges, neoplasm of unknown behavior of the meninges of unspecified, neoplasm of unknown brain behavior, Neoplasm of unknown behavior of brain, subtendium, neoplasm of unknown behavior of brain, neoplasm of unknown behavior of brain, neoplasm of unknown behavior of cranial nerve, neoplasm of unknown behavior of spinal cord, Neoplasm of unknown behavior of central nervous system, neoplasm of unknown behavior of endocrine gland, neoplasm of thyroid gland of unknown behavior, neoplasm of unknown behavior of adrenal gland, neoplasm of unknown behavior of adrenal gland unspecified Neoplasm of unknown behavior, neoplasm of unknown behavior of pituitary gland, neoplasm of unknown behavior of craniopharyngeal tube, neoplasm of unknown behavior of pineal gland, neoplasm of unknown behavior of carotid body, aortic body and others Neoplasm of unspecified behavior of paraganglion, neoplasm of unspecified behavior of endocrine gland, polycythemia vera, myelodysplastic syndrome, refractory anemia without ring iron blasts, specified as Refractory anemia, refractory anemia with excess blast cells [RAEB], unspecified myelodysplastic syndrome, lymphoid, hematopoietic tissue unknown Other neoplasms, histiocytic and mast cell tumors of unknown behavior, chronic myeloproliferative disease, monoclonal gamma pathology, essential (hemorrhagic) thrombocytopenia, myelofibrosis, lymphoma, other neoplasms of unknown hematopoietic tissue Organism, lymphoma, unspecified, neoplasm of unknown hematopoietic behavior, neoplasm of unknown behavior in other and unspecified sites, neoplasm of unknown bone/articular cartilage behavior, connective/soft tissue behavior unknown Neoplasm of unknown behavior of peripheral nerves and autonomic nervous system, neoplasm of unknown behavior of retroperitoneum, neoplasm of unknown behavior of peritoneum, neoplasm of unknown behavior of skin, unknown behavior of breast Neoplasm of unknown behavior of digestive system, neoplasm of unknown behavior of respiratory system, neoplasm of unknown behavior of bone, soft tissue and skin, neoplasm of unknown behavior of breast, bladder of unknown behavior Neoplasms, other genitourinary organs, neoplasms of unknown behavior, kidneys, neoplasms of unknown behavior, other GU organs neoplasms of unknown behavior, neoplasms of unknown behavior in brain, glands and other parts of the nervous system Neoplasm of unknown behavior of the retina, neoplasm of unknown behavior of retina and choroid, or neoplasm of unknown behavior of unspecified site.

In some embodiments of the present disclosure, the cancer may not be one of the aforementioned cancers.

In some preferred embodiments of the present disclosure, the cancer may be selected from the group consisting of: lymphoma, squamous cell carcinoma (eg epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung; peritoneal cancer; hepatocellular carcinoma; stomach or abdominal cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver tumor; breast cancer; colorectal cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary adenocarcinoma; kidney or kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocarcinoma; anal carcinoma; penile carcinoma; and head and neck cancer. In some particularly preferred embodiments of the present disclosure the cancer may be lymphoma. In a more particularly preferred embodiment of the present disclosure the cancer may be a B cell lymphoma or a T cell lymphoma. In some particularly preferred embodiments of the present disclosure the cancer may be a non-Hodgkin's lymphoma. In another preferred embodiment, the cancer may be a post-transplant lymphoproliferative disorder. In some other particularly preferred embodiments of the present disclosure, the cancer may be a solid tumor cancer.

In embodiments where the methods of the present disclosure are performed on a subject having, suspected, or ever diagnosed with cancer, the NKT cells, T cells, and/or dendritic cells produced by these methods are capable of treating cancer. have. In this context, "treat" means exerting a beneficial therapeutic effect in a subject, which may be any overall clinical benefit resulting from the methods of the present disclosure. These overall clinical benefits include, for example: prolonged survival, partial or complete disease remission (eg, as assessed by % myeloblasts and/or normal maturation of cell lines), slowing or absence of disease progression (eg, For example, as assessed by change in % myeloblasts), tumor shrinkage (eg, a reduction of at least 5, 10, 20, 30, 40% of tumor volume), reduction in tumor burden (eg, tumor burden) reduction of more than 5, 10, 20, 30, 40%), slowing or absence of tumor enlargement, slowing or absence of increased tumor burden, improving quality of life (e.g., as in the Functional Assessment of Cancer Therapy (FACT) questionnaire) As assessed by health-related quality of life questionnaire), progression-free survival, overall survival, hematological improvement (eg: increased blood hemoglobin, platelet count and/or neutrophil count), bone marrow response (eg: myeloblasts) bone marrow less than 5%; reduction of more than 30%, 40%, 50% in myeloblasts; circulating myeloblasts with Hour rods and absence of myeloblasts; absence of extramedullary disease), hematological recovery (e.g.: >11 g/dL hemoglobin, ≥100x109/L platelets, and/or ≥1x109/L peripheral blood neutrophils), negative response to genetic markers (eg, CEBPA, NPM1, or FLT3), or any other positive patient It could be one of the results.

The overall clinical benefit may be an “anti-tumor effect”. As used herein, an “anti-tumor effect” refers to a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, a decrease in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or Refers to the biological effect that can be exhibited by the improvement of various physiological symptoms associated with tumors. An anti-tumor effect may also refer to the prevention of tumor development, eg, a vaccine. For example, computed tomography (CT), or magnetic resonance imaging (MRI) imaging techniques; X-ray imaging, such as mammography; ultrasound imaging; nuclear imaging such as positron emission tomography (PET), PET/CT scan, bone scan, gallium scan, or metaiodobenzylguanidine (MIBG) scan; bioluminescence imaging (BLI); fluorescence imaging (FLI); Appropriate methods for measuring tumor volume/burden using BD ToF (infrared-based 3D time-of-flight camera) imaging are well known to those skilled in the art.

Thus, in some embodiments, the NKT cells of the present disclosure are capable of treating cancer through tumor infiltration. In some embodiments, the NKT cells of the present disclosure are capable of treating cancer through the release of immune activating cytokines. In some embodiments, the NKT cells of the present disclosure are capable of engulfing and killing cancer cells in a subject. In some embodiments, the NKT cells of the present disclosure promote infiltration of other immune cells into the tumor. In some embodiments, the NKT cells of the present disclosure directly kill cancer cells via CD1d-induced apoptosis.

In some embodiments, the T cells of the present disclosure are capable of treating cancer through tumor infiltration. In some embodiments, the T cells of the present disclosure are capable of treating cancer through the release of immune activating cytokines. In some embodiments, the T cells of the present disclosure promote infiltration of other immune cells into the tumor. In some embodiments, T cells of the present disclosure directly kill cancer cells by inducing apoptosis by expressing a ligand that binds to a death receptor on a target cell. In some embodiments, T cells of the present disclosure are capable of ingesting or swallowing cancer cells in a subject. In some embodiments, T cells are capable of secreting cytotoxic molecules that kill cancer cells.

In some embodiments, the dendritic cells of the present disclosure are capable of treating cancer through immune surveillance. Dendritic cells (DCs) are antigen-presenting cells derived from bone marrow precursors and form a widely distributed cellular system throughout the body. DCs exert a late activation of naive T lymphocytes that elicit immune surveillance and diverse immunological responses to exogenous and endogenous antigens. DCs are sentinel cells responsible for pathogen recognition and tissue damage signaling, which induce migration to lymphoid organs, where they carry out the activation of T, natural killer (NK), NKT, and other subsets of B lymphocytes. Mature phenotypic cDCs are characterized by increases in MHCII, CD80, CD86, and CD40. In some embodiments, dendritic cells of the present disclosure promote infiltration of other immune cells, such as T cells, into the tumor. In some embodiments the dendritic cells of the present disclosure enhance the T cell response to cancer by presenting the cancer antigen to the T cells. In some embodiments, the dendritic cells of the present disclosure can directly kill cancer cells by inducing apoptosis by expressing a ligand that binds to a death receptor on a target cell.

As used herein, “autoimmune disease” refers to autoimmune disorders and other diseases that result from abnormal immunity in which the immune system abnormally attacks a subject's own components. (In healthy subjects, the immune system establishes resistance to the subject's own components, preventing damage to the autoimmune response). Examples of various autoimmune diseases are described herein and include, but are not limited to, celiac disease, diabetes type 1, Graves' disease, inflammatory bowel disease, transient osteoporosis, multiple sclerosis, psoriasis, rheumatoid arthritis and systemic lupus erythematosus.

Autoreactive immune cells bind to stressed cells and microorganisms such as mycobacteria , Escherichia coli , and Plasmodium, particularly (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). It expresses high levels of phosphate antigen, a diphosphate-containing metabolite, as well as phosphate antigen produced by Humans do not produce HMB-PP. However, most Gram-negative bacteria, including Mycobacterium tuberculosis, Mycobacterium bobus, Clostridium difficile, Listeria monocytogenes, malaria parasites, and Toxoplasma gondii and Kistosoma japonicum. creates it Gamma delta T cells/receptors are highly sensitive to HMB-PP, zoledronate and isopentyl pyrophosphate (IPP), mycorillarabinogalactan peptidoglycan (mAGP), and iso-butylamine (IBA). . Butyrophilin family members such as BTN2A1, BTN3A1, BTNL3, BTNL8, BTNL1, BTNL6, Skint1 and Skint2 play an important role in gamma delta T cell recognition of phosphorescent antigens. Aminobisphosphonate stimulation of peripheral blood mononuclear cells (PBMCs) can also activate gamma delta T cell receptors. IL-18 can enhance the response of gamma delta T cell receptors to phosphorus antigens.

In some embodiments of the present disclosure the autoimmune disease may be: allergy, asthma, graft-versus-host disease (GvHD), steroid resistant GvHD, achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, protozoa Alopecia, alopecia, transient osteoporosis, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune autonomic dystrophy, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED) ), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Valero's disease, Behcet's disease, benign mucosal pemphigus, pemphigus bullae, castle Chronic (CD), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA)), epileptic pemphigus , Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis herpetiformis, dermatomyositis, Debick's disease (optic neuromyelitis), disc lupus, Dressler's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, multiple Granulomatosis with vasculitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schoonrein purpura (HSP), herpes gestational or pemphigus gestational erythematosus (PG), pyelonephritis (HS) (Acnein bursa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerotic disease, immune thrombocytopenia (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), Juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukoblastic vasculitis, lichen planus, lichen planus, ligamentous conjunctivitis, linear IgA disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Muren's ulcer, Mucha-Haberman's disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis , myasthenia gravis, myositis, narcolepsy, neonatal lupus, optic neuromyelitis, neutropenia, ocular macular pemphigus, optic neuritis, palindromic rheumatism (PR), PANDAS, neoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), paroxysmal Romberg syndrome, Pars flatulitis (peripheral uveitis), Pasonage-Turner syndrome, pemphigus, peripheral neuropathy, peripheral encephalitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyline syndrome types I, II, III, Polymyalgia rheumatoid arthritis, polymyositis, post-myocardial infarction syndrome, pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red blood cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive Arthritis, reflex sympathetic dystrophy, recurrent polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmune, spasticity In Syndrome (SPS), Subacute Bacterial Endocarditis (SBE), Susak Syndrome, Sympathetic Ophthalmitis (SO), Takayasu Arteritis, Temporal Arteritis/Giant Cell Arteritis, Thrombocytopenic Purpura (TTP), Tolosa Hunt Syndrome (THS) , transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Bogt-Koyanagi-Harada disease, hemophagocytic lymphohistiocytosis, multiple myeloma, Allergen-specific immunotherapy, autosomal dominant haploid dysfunction, anterior interosseous nerve syndrome, Chug-Strauss syndrome, systemic vasculitis, chronic graft-versus-host disease, myoclonus-myoclonus syndrome, necrotizing autoimmune myopathy (NAM), lung Sarcoma Carcinoma, Waldenstrom's Macroglobulinemia (WM), Bull Lyme, Behcet's disease, alopecia areata (AA), acute-chronic liver failure, melanoma, 'organized bronchiolitis syndrome', or encephalitis. In some embodiments the autoimmune disease may be: rheumatoid arthritis, rheumatoid fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma , polymyositis/scleroderma, polymyositis/dermatomyositis, ulcerative proctitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, telangiectasia, seasonal allergy, perennial allergy, food allergy, anaphylaxis, mastocytosis, allergic rhinitis, Atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenomegaly, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, autoimmune lymphoproliferative syndrome , Biscott-Aldrich syndrome, chronic granulomatosis, common variable immunodeficiency syndrome (CVID), hyperimmunoglobulin E syndrome, Hashimoto's thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sidnum chorea, myasthenia gravis , multiple gland syndrome, pemphigus bullosa, Henoch-Schoonrein purpura, streptococcal purpura, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, good Pascher's syndrome, thrombotic purpura, Sjogren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, hyperthyroidism, chronic active hepatitis, polychondritis, pemphigus vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, spinal cord, Giant cell arteritis,/polymyalgia, congestive anemia, rapidly progressive glomerulonephritis, psoriasis, fibrosing alveolitis, or cancer.

In some embodiments of the present disclosure, the autoimmune disease may not be one of the aforementioned autoimmune diseases.

In some preferred embodiments of the present disclosure, the autoimmune disease may be selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes mellitus (T1D), scleroderma, pemphigus, and lupus. . In some other preferred embodiments of the present disclosure the autoimmune disease may be selected from the group consisting of: graft-versus-host disease (GvHD), and allergic disorders such as asthma. In some particularly preferred embodiments of the present disclosure the autoimmune disease may be type 1 diabetes mellitus (T1D).

In embodiments where the methods of the present disclosure are performed on a subject having, suspected of having, or diagnosed with an autoimmune disease, the NKT cells, T cells, and/or dendritic cells produced by these methods Cells can treat autoimmune diseases. In this context, "treat" means exerting a beneficial therapeutic effect in a subject, which may be any overall clinical benefit resulting from the methods of the present disclosure. This overall clinical benefit can be, for example, one of the following: reduced fatigue, reduced muscle pain, reduced edema and redness, reduced low fever, reduced concentration, reduced numbness and numbness in limbs and limbs, reduced urination, reduced hair loss, skin rash Reduces, restores normal blood sugar, increases C-peptide, improves wound healing, reduces diarrhea, reduces muscle cramps, improves muscle tone and control, reduces skin rashes or skin discoloration, improves weight maintenance, reduces muscle or joint pain, improves digestive tract comfort, normal heart rate , decreased anxiety, decreased Expanded Disability Status Scale (EDSS) scores, and decreased intrinsic active lesions in the brain as measured by gadolinium-enhanced MRI.

In some embodiments, the NKT cells of the present disclosure are capable of directly killing autoreactive T and/or B lymphocytes, increasing the Treg:T lymphocyte ratio, inhibiting the activity of autoreactive T and/or B lymphocytes, reducing inflammation, or autoreactive lymphocytes. It is possible to treat autoimmune diseases by reducing the trafficking of

In some embodiments, the T cells of the present disclosure include direct killing of autoreactive T and/or B lymphocytes, increased Treg : T lymphocyte ratio, inhibition of activity of autoreactive T and/or B lymphocytes, reduced inflammation, or autoreactive lymphocytes. It is possible to treat autoimmune diseases by reducing the trafficking of

In some embodiments, the dendritic cells of the present disclosure are capable of treating an autoimmune disease through the release of immune activating cytokines or by promoting T cell death of autoreactive T and/or B lymphocytes.

As used herein, “infectious disease” (or “microbial disease”) refers to a disease or disorder resulting from infection of a subject's body by an infectious agent (pathogen), such as a virus, bacteria, or fungus. In some embodiments of the present disclosure the infectious disease may be: Acinetobacter infection (Acinetobacter baumani), actinomycosis (Actinomyces israeli, Actinomyces gerenceriai, and Propionibacterium propioni) Onycus) African Sleeping Disease or African Trypanosomiasis (Trypanosoma brusei), AIDS (Acquired Immunodeficiency Syndrome) (Human Immunodeficiency Virus), Amebosis (Entamoeba histolytica), Anaplasmosis (Anaplasma spp.), Eye Geostrongilosis (angiostrongilus), anisakisis (anisakis), anthrax (Bacillus anthracis), Arcanobacterium haemolyticum infection (Arcanobacterium haemolyticum), Argentine hemorrhagic fever (Junin virus) , Roundworm (Ascaris lumbricoid), Aspergillosis (Aspergillus spp.), Astroviral infection (Astroviral spp.), Babesiosis (Babesia spp.), Bacillus cereus infection (Bacillus cereus) ), bacterial pneumonia (multi-bacterial), bacterial vaginosis (list of bacterial vaginosis microflora), Bacteroides infection (Bacteroides spp.), valantidiosis (Valantidium coli), bartonellosis (Bartonella), Weil Lijascaris infection (Bylizascaris spp.), BK virus infection (BK virus), black piedra (Piedra orte), blastocystosis (blastocystis spp.), blastomyosis (Blastomyces dermatitis) dis), Bolivian hemorrhagic fever (Machupo virus), botulism (and infant botulism) (Clostridium botulinum; Note: botulism is caused by ingestion of botulinum toxin, not infection by Clostridium botulinum), Brazil Hemorrhagic fever (Sabia virus), Brucellosis (Brusella spp.), Lymph node plague (Enterobacteriaceae), Burkholderia infection, usually Burkholderia cepacia and other Burkholderia spp., Buruli ulcer (Mycobacterium ulceae) lance), calicivirus infections (noroviruses and sapoviruses) (caliciviridae), campylobacteriasis (Campylobacter spp.), candidiasis (moniliosis; vaginitis) (commonly Candida) albicans and other Candida species), nematodes nematodes (intestinal disease caused by Capillaria philippinesis, liver disease caused by Capillaria hepatica and lung disease caused by Capillaria aerophila), carion disease (Bartonella bacilliformis) ), feline scratching disease (Bartonella hensellae), cellulitis (commonly group A streptococcus and staphylococcus), Chagas disease (trypanosomiasis in the United States) (trypanosoma cruzi), chancroid (Haemophilus ducreyi), Chickenpox (Varicella Zoster Virus (VZV)), Chigunya (Alphavirus), Chlamydia (Chlamydia trachomatis), Chlamydophila pneumoniae infection (Taiwan Acute Respiratory Agent or TWAR) (Chlamydophila pneumoniae), Cholera (Vibrio cholera), chromoblastomycosis (commonly Fonsecae pedrosois), leukomycosis (Batracochytrium dendravatidis), hepatochondriasis (Clonokis sinensis), Clostridium difficile colitis (Clo. Stridium difficile), Coccidioidomycosis (Coccidioidiz imitis and Coccidioidiz posadasi), Colorado tick fever (CTF) (Colorado tick fever virus (CTFV)), Cold (acute viral nasopharyngitis) ; cold) (usually rhinoviruses and coronaviruses), coronavirus, Creutzfeldt-Jakob disease (CJD) (PRNP), Crimea-Congo hemorrhagic fever (CCHF) (Crimea-Congo hemorrhagic fever virus), yeastomycosis (Cryptococcus neopor) mans), Cryptosporidium (Cryptosporidium spp.), cutaneous larval migration (CLM) (generally Ancilostoma brasiliense; several other parasites), Cystosporidiosis (Cyclospora calletanensis), Cystosporidiosis ( tania solium), cytomegalovirus infection (cytomegalovirus), dengue fever (dengue virus (DEN-1, DEN-2, DEN-3 and DEN-4) - flavivirus), desmodemus infection (green algae desmode moose armatus), dientamebosis (Dienthamoeba fragilis), diphtheria corynebacterium diphtheria), diphylobotrosis (diphylobotrium), medinaemia (Dracunculus medinensis), Ebola Hemorrhagic Fever (Ebolavirus (EBOV)), Echinococcus spp. , enterovirus infection (enterovirus spp.), typhoid (Rikecha provachecki), infectious erythema (5th disease) (parvovirus B19), breakthrough (6th disease) (human herpesvirus 6 (HHV-6) and Human Herpesvirus 7 (HHV-7)), Fasciola Hepatica and Fasciola zycantica), Hypertrophosis (Fasciolopsis bursky), Fatal Familial Insomnia (FFI) (PRNP) , Onchocerciasis (Filarioidea superfamily), Food poisoning caused by Clostridium perfringens (Clostridium perfringens), free-living amoeba infection (multiple), Fusobacterium infection (Fusobacterium spp.), gas gangrene ( Clostridial muscle necrosis) (usually Clostridium perfringens; other Clostridial species), geotricumosis (Giotricum candidum), Gerstmann-Streusler-Scheinker disease (GSS) (PRNP) , giardiasis (Giadia lamblia) paralytic (Burkholderia malaysia), hookworm (Ganatostoma spinigerum and Ganatostoma hispidum), gonorrhea (Niceria gonorhoae), inguinal granuloma (donovaniosis) (Krebsiella granuloma), group A streptococcal infection (Streptococcus pyogenes) , Group B Streptococcus infection (Streptococcus agalactiae), Haemophilus influenzae infection (Hamophilus influenzae) Hand, foot and mouth disease (HFMD) (enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71)), Hantavirus pulmonary syndrome (HPS) (Sinnombre virus), Heartland virus disease (Heartland virus), Helicobacter pylori infection (Helicobacter pylori), hemolytic uremic syndrome (HUS), E. coli O157:H7, O111 and O104:H4, Nephrotic Syndrome Hemorrhagic Fever (HFRS) ) (Buniaviridae), hepatitis A (hepatitis A virus), hepatitis B (hepatitis B virus), hepatitis C (hepatitis C virus), hepatitis D (hepatitis D virus), hepatitis E (Hepatitis E virus), Herpes simplex (Herpes simplex virus 1 and 2 (HSV-1 and HSV-2)), Histoplasmosis (Hitoplasma capsulatum), Hookworm infection (Ancilostoma duodenale and Neca) Thor americanus), Human Bocavirus Infection (Human Bocavirus (HBoV)), Human Ewingi Ehrlichosis (Ehrlicia Ewingi), Human Granulocyte Anaplasmosis (HGA) (Anaplasma phagocytophilium), Human Meta Pneumovirus infection, human metapneumovirus (hMPV), human mononuclear ehrlichiosis (Ehrlicia chaffeensis), human papillomavirus (HPV) infection (human papillomavirus (HPV)), human parainfluenza virus infection (human parainfluenza virus (HPIV) )), oncotic dwarfism (H. nana and Hymenorepsis diminuta), Epstein-Barr virus infectious mononucleosis (single) (Epstein-Barr virus (EBV)), Influenza (flu) (Orthomyxoviridae) Sporidiosis (iso pora valley), Kawasaki disease (unknown; Evidence shows that it is contagious) keratitis (multiple), Kingella kingae infection (kingella kingae), kuru disease (PRNP), Lassa fever (Lassa virus), Legionellosis (Legionella disease) (Legionella pneumophila), Legionellosis ( Pontiac fever) (Legionella pneumophila), leishmaniasis (Leishmania spp.), leprosy (Mycobacterium repre and Mycobacterium lepromatosis), leptospirosis (leptospira spp.), listeriosis (Listeria monocytosis) Genes), Lyme disease (Lyme borreliosis) (Borrelia burgdorferi, Borrelia garini, and Borrelia apjeli), Lymphofibrosis (epithelial disease) (Uchereria vancrofti and Brussia Malay), lymphocytic choriomeningitis (Lymphocytic Choriomeningitis Virus (LCMV)), Malaria (Psmodium spp.), Marburg Hemorrhagic Fever (MHF) (Marburg Virus), Measles (Measles Virus), Middle East Respiratory Syndrome (MERS) (Middle East Respiratory Syndrome Coronavirus) ), melioidosis (Whitmore's disease) (Burkholderia pseudomalei), meningitis (multiple), meningococcal disease (Nisseria meningitidis), yokogawa schizophrenia (commonly metagonimus yokagawai), microsporidiosis Pneumoconiosis (microcytoplasm), molluscum contagious (MC) (Moluscum contagiosum virus (MCV)), monkeypox (monkey pox virus), mumps (mumps virus), typhus (typhoid endemic) (rickettsia typhi) , Mycoplasma pneumoniae (Mycoplasma pneumoniae), Mycoplasma (clarified) (multiple species of bacteria (actinomycetes) and fungi (mycobacteriaceae)), parasitic fly larvae), neonatal conjunctivitis (neonatal blepharitis) (most common Chlamydia trachomatis and Neisseria gonorrhea), norovirus (children and infants) new) variant Creutzfeldt-Jakob disease (vCJD, nvCJD), PRNP), nocardiasis (usually Nocardia asteroides and other nocardia spp.), onchocerciasis (river blindness) (oncocirca bolburus), liver flukeosis (officetorchis biberini and officetorchis pelinus), paracoccidioidomycosis (South American blastomycosis) (paracoccus) cd's zbrasiliensis), pulmonary schizophrenia (usually Paragonimus westermani and other Paragonimus spp.), Pasteurella disease (Pasteurella spp.), claustrophobia (head lice) (Pediculus humanus capitis), beadism ( body) (Pediculus humanus corpus), pubic lice (Pubic lice, gay) (Ptyrus pubis), pelvic inflammatory disease (PID) (multiple), whooping cough (pertussis) (Bordetella pertussis) , plague (Yersinia pestis), pneumococcal infection (Streptococcus pneumoniae), pneumococcal pneumonia (PCP) (pneumocystis giroveci), pneumonia (multiple), polio (poliovirus), pre Botella infection (Prevotella spp.), primary amoebic meningoencephalitis (PAM) (commonly negleria fowleri), progressive multifocal leukoencephalopathy (JC virus), parakeet disease (Chlamydophila picitasi), Q fever cox ella verneti), rabies (rabies virus), relapsing fever (Borrelia hermici, Borrelia licerentis, and other Borrelia spp.), respiratory syncytial virus infection (respiratory syncytial virus (RSV)), linospor Lycheidosis (Rinosporidium severi), Rhinovirus infection (Rinovirus), Rickettsia infection (Rikecha spp.), Rickettsia dou (Rikecha akari), Rift Valley fever (RVF) (Rift Valley fever virus), Rocky Mountain Spot Fever ( RMSF) (Rikecha ricketts), rotavirus infection (rotavirus), rubella (rubella virus), salmonellosis (Salmonella spp.), SARS severe acute respiratory syndrome) (SARS coronavirus), scabies (Sarcoptes scabiei), Schistosomiasis (schistosoma spp.), sepsis (multiple), shigella spp. (bacterial dysentery) (shigella spp.), herpes zoster (herpes zoster) (varicella zoster virus (VZV)), smallpox (smallpox) (varicella or smallpox), sporotrixosis (Sporotrix shenki), staphylococcal food poisoning (staphylococcal spp.), staphylococcal infection (staphylococcal spp.), hepatoma (Strongiloid stecoralis), subacute sclerosing panencephalitis ( measles virus), syphilis (Treponema pallidium), Onchocerciasis (Tannia species) , tetanus (clostridium tenani), tinea tinea ganglia (barber pruritus) (commonly trichophyton species), tinea cap (tinea scalp) (usually trichophyton tonic), tinea corpuscle (tinea body tinea) ) (usually Trichophyton species), tinea pubis (choke) (usually epidermophyton floccosome, trichophyton rubrum, and trichophyton mentagrophyte), tinea (ringworm of the hand) (trichophyton rubrum), black Ringworm (commonly Ortea Bernecki), Ringworm (Athlete's Foot) (Commonly Trichophyton spp.), Ringworm Onychomycosis (Onychomycosis) (usually Trichophyton spp.), Trichophyton spp. , Toxocarasis (Larvae Ocular Dystrophy (OLM)) (Toxocara canis or Toxocara katii), toxocarasis (Lucifera larvae (VLM)) (Toxocara canis or Toxocara kati), trachoma (chlamydia) trachomatis), toxoplasmosis (Toxoplasma gondii), trichinosis (Tricinella spiralis), trichomoniasis (Trichomonas virginalis), whipworm ( whipworm infection) (Trichuris trichiura), tuberculosis rosacea (commonly Mycobacterium tuberculosis), tularemia (Francisella tularensis), typhoid fever (Salmonella enterica subspecies enterica, serotype typhoid), typhoid fever (ikesia), ureaplasma urea lichenicum infection (Ureaplasma urealyticum), valley fever (Coccidioidiz imitis or Coccidioidiz posadasi), Venezuelan equine encephalitis (Venezuelan equine encephalitis virus), Venezuelan hemorrhagic fever (Guanaritovirus), Vibrio bull nipicus infection (Vibrio vulnipicus), Vibrio parahaemolytic enteritis (Vibrio parahaemolyticus), viral pneumonia (multiple virus), West Nile fever (West Nile virus), leukoplakia (ringworm) (tricosporon bay) geli), Yernicia pseudotuberculosis infection (Yernicia pseudotuberculosis), arsiniasis (Ersinia enterocolica), yellow fever (yellow fever virus), zygotomycosis (Mucoral order (Mo. Mycosis) and Insomycetes (Insectomycosis)) Human Immunodeficiency Virus [HI] V] HIV disease with diseases, infections and parasitic diseases, HIV disease with mycobacterial infection, HIV disease with cytomegalovirus disease, HIV disease with other viral diseases, HIV disease with candidiasis, other mycoses HIV disease with cystic cystic pneumonia HIV disease with malignant neoplasm, HIV disease with Kaposi's sarcoma, HIV disease with Burkitt's lymphoma, HIV with other types of non-Hodgkin's lymphoma disease, HIV disease with other malignant lymphocytes, hematopoietic and related tissue neoplasms, HIV disease with multiple malignant neoplasms, HIV disease with other malignant neoplasms, HIV disease with unspecified malignant neoplasms; HIV disease with encephalopathy, HIV disease with lymphocytic interstitial pneumonia, HIV disease with wasting syndrome, HIV disease with multiple diseases classified elsewhere, HIV disease with other conditions, HIV disease acute HIV infection syndrome, HIV disease with (persistent) systemic lymphadenopathy, HIV disease with hematologic and immunological abnormalities, HIV disease with certain other conditions, or HIV disease, unspecified. In some embodiments of the present disclosure the infectious disease may be an infection by a virus, such as a virus of one of the following viridae: a) adenovirae, eg, adenovirus species; b) herpesviruses such as herpes simplex type 1, herpes simplex type 2, varicella zoster virus, Epstein-Barr virus, human cytomegalovirus, human herpesvirus type 8 species; c) papillomaviruses, for example human papillomavirus species; d) polyomaviridae, eg BK virus, JC virus species; e) poxviridae, eg smallpox species: f) hepadnaviridae, eg hepatitis B virus species: g) parvovirus family, eg human bocavirus, parvovirus B19 species; h) Astroviridae, eg human astroviral species: i) Caliciviridae, eg Norwalk virus species; j) Flaviviridae, eg hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae, eg rubella virus species; 1) Hepeviridae, for example hepatitis E virus species; m) retroviridae, for example human immunodeficiency virus (HIV) species; n) Orthomyxoviridae, eg influenza virus species; o) arenaviridae, eg, guanaritovirus, hunin virus, lasa virus, machupo virus, and/or sabia virus species; p) Bupuriviridae, eg Crimea-Congo hemorrhagic fever virus species; q) filoviridae, eg Ebola virus and/or Marburg virus species; Paramyxoviridae, eg, measles virus, mumps virus, parainfluenza virus, respiratory syncytial virus, human metapneumovirus, Hendra virus and/or nipa virus species; r) Rhabdoviridae, for example rabies virus species; s) Reoviridae, for example rotavirus, orbivirus, cortivirus and/or barnavirus species.

In some embodiments of the present disclosure, the infectious disease may not be one of the aforementioned infectious diseases.

In some embodiments, the infectious disease may be a disease caused by infection with the influenza A (flu A) virus. In some embodiments the influenza virus is a pandemic influenza virus of avian or porcine origin, e.g., H5N1, H7N3, H7N7, H7N9 and H9N2 (avian subtype) or H1N1, H1N2, H2N1, H3N1, H3N2, or H2N3 (porcine subtype) ) can be

In some preferred embodiments of the present disclosure, the infectious disease may be HIV, such as residual HIV disease, herpes, hepatitis or human papillomavirus. In another preferred embodiment, the infectious disease may be a disease caused by infection by a coronavirus, for example COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, a disease caused by SARS-CoV-2) have.

In embodiments where the methods of the present disclosure are performed on a subject having, suspected of having, or diagnosed with an infectious disease, the NKT cells, T cells, and/or dendritic cells produced by such methods are capable of treating the infectious disease. can be treated In this context, "treat" means exerting a beneficial therapeutic effect in a subject, which may be any overall clinical benefit resulting from the methods of the present disclosure. This overall clinical benefit could be, for example, one of the following: reduced fever, reduced diarrhea, less cough, less muscle pain, less fatigue, less CRP, less time on ventilator, less need for additional oxygen, less organ damage after recovery.

In some embodiments, the NKT cells of the present disclosure engulf and kill infectious organisms, activate other innate and adaptive immune cells, recruit other immune cells to the site of infection (eg, an organ infected by a virus), and Infectious diseases can be treated by depleting infected immune cells (eg, monocytes activated by COVID-19).

In some embodiments, the T cells of the present disclosure are capable of treating infectious diseases through the release of immune activating cytokines. In some embodiments, T cells of the present disclosure are capable of treating infectious diseases through the release of cytokines (eg, TNF-alpha, IFN-gamma) that have anti-microbial or anti-viral effects. In some embodiments, T cells of the present disclosure are capable of treating an infectious disease by inducing apoptosis by expressing a ligand that binds a death receptor on a target cell. In some embodiments, the T cell is capable of secreting a cytotoxic molecule that kills an infectious organism. In some embodiments, T cells of the present disclosure are capable of ingesting or swallowing an infectious organism.

In some embodiments, dendritic cells of the present disclosure are capable of treating infectious diseases by transducing pathogen-associated signals to the adaptive portion of the immune system. In some embodiments, the dendritic cells of the present disclosure can treat an infectious disease by priming cytotoxic T cells to kill the infectious organism and/or promote T cell infiltration into the site of infection.

In embodiments where the infectious disease is a disease caused by infection by a coronavirus, e.g., COVID-19, the NKT cells of the present disclosure engulf and kill the coronavirus and/or activate other innate and adaptive immune cells to treat the disease. can be treated

Accordingly, the present disclosure also provides a method of treating a disease due to a coronavirus infection in a subject, the method comprising administering to the subject a glucocorticoid receptor (GR) modulator at an approximately at least 6 mg/kg human equivalent dose of dexamethasone base (HED) ) and administering at an equivalent dose. In some embodiments, the glucocorticoid receptor (GR) modulator may be a glucocorticoid, preferably dexamethasone or betamethasone. In some embodiments, the glucocorticoid receptor (GR) modulator may be administered at a dose equivalent to approximately at least 15 mg/kg human equivalent dose (HED) of dexamethasone base. In some preferred embodiments, the glucocorticoid receptor (GR) modulator may be administered at a dose equivalent to about 18 mg/kg to 30 mg/kg human equivalent dose (HED) of dexamethasone base. In some preferred embodiments, the disease is COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, disease caused by SARS-CoV-2) or SARS-CoV or MERS. In some embodiments, the glucocorticoid receptor (GR) modulator induces a population of NKT cells and/or T cells as disclosed elsewhere herein. In some embodiments, the glucocorticoid receptor (GR) modulator activates a population of dendritic cells as disclosed elsewhere herein.

In some preferred embodiments, the present disclosure provides a method of treating COVID-19 (coronavirus 2019; severe acute respiratory syndrome coronavirus 2, a disease caused by SARS-CoV-2) in a subject, the method comprising: administering to the subject dexamethasone or betamethasone at a dose equivalent to about 15 mg/kg to 30 mg/kg human equivalent dose (HED) of dexamethasone base.

In embodiments where the disease is due to infection by a coronavirus, eg, COVID-19, the glucocorticoid receptor modulator may be administered in combination with a proton pump inhibitor (eg omeprazole) and/or hydrocortisone. In this context, "in combination with" may mean simultaneous administration or may mean separate and/or sequential administration in any order.

In some embodiments of the methods of the present disclosure, a method of producing/mobilizing a population of natural killer T cells (NKT cells), a method of producing/mobilizing a population of T cells, and/or recruiting/activating a population of dendritic cells The method may further comprise isolating NKT cells, T cells, and/or dendritic cells, or a population of NKT cells, T cells, and/or dendritic cells from the subject or from a sample derived from the subject. Accordingly, the present disclosure provides isolated NKT cells isolated T cells, and isolated dendritic cells, as well as isolated populations of NKT cells, T cells, and dendritic cells. Isolated cells and isolated cell populations can be characterized by the pattern of surface proteins they express as outlined above.

Appropriate methods for isolating cells and cell populations from mixed samples are well known to those skilled in the art - for example flow sorting (e.g. fluorescence-activated cell sorting; FACS) and magnetic particle sorting (e.g. magnetic-activated cell sorting) ; MACS), microfluidic cell sorting, density gradient centrifugation, immunodensity cell isolation, expansion of cell cultures based on growth factors and other components of the medium. In some preferred embodiments of the present disclosure, the isolating step is performed by fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS).

In embodiments in which the NKT cells, T cells, and/or dendritic cells are isolated from a sample derived from the subject, the sample is selected from blood, plasma, tumor biopsy or surgically removed tumor, bone marrow, liver, spleen biopsy and adipose or may be selected from the group consisting of adipose tissue.

In some embodiments, the isolating step can be performed at least about 1, 3, 12, 24, 48, 72, 96, 120, 144, or 168 hours after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator. In some embodiments, the isolating step can be performed at least about 1, 3, 8, 9, 10, 11, 12, 13, 14, or 15 days after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator. In some preferred embodiments, the isolating step is performed at least about 48 hours after said administration. In some other preferred embodiments, the isolating step is performed about 1, 3, or 48 hours after said administration. In some embodiments, the isolating step comprises about 1, 3, or 48 hours to 13 days, about 1, 3, or 48 hours to 168 hours, about 1, 3, after administration of the glucocorticoid receptor (GR) modulator or ICAM3 modulator, or from 48 hours to 120 hours, from about 1, 3, or from 48 hours to 96 hours, or from about 1, 3, or from 48 hours to 72 hours. In some preferred embodiments, the isolating step is performed about 1, 3, or 48 to 72 hours after said administration. In some embodiments, the isolating step can be performed within 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours after administration of the glucocorticoid. In some preferred embodiments, the isolation step can be performed within 3 hours after administration of the glucocorticoid. In some particularly preferred embodiments the isolating step may be performed within one hour after administration of the glucocorticoid. In some preferred embodiments, wherein the subject suffers from cancer, an infectious disease or an autoimmune disease, isolating the NKT cells is performed on a blood sample of the subject within 3 hours after administration of the glucocorticoid, preferably within 1 hour after administration of the glucocorticoid. can be performed.

In some preferred embodiments of methods comprising an isolating step, the subject may be a healthy subject, such as a healthy adult human subject. A healthy subject in this context is a subject that does not have the disease.

The isolated NKT cells, T cells, and/or dendritic cells of the present disclosure, and the isolated NKT cell populations, T cell populations, and/or dendritic cell populations can be expanded in culture. Methods and reagents suitable for cell culture and expansion are well known to those skilled in the art. For example, long-term incubation with IL-2, soluble anti-CD28 antibody, anti-CD3 epsilon antibody, anti-TCRbeta antibody, and glycolipids such as KRN7000, PBS44, or PBS57 has been shown to produce robust expansion of NKT cells. (Watarai et al 2008, incorporated herein by reference in its entirety). Accordingly, in some embodiments of the methods of the present disclosure, the method of generating a population of natural killer T cells (NKT cells), a method of generating a population of T cells, and/or activating the population of dendritic cells comprises the steps of: It may further comprise the step of expanding the isolated NKT cells, T cells, dendritic cells or NKT cells, T cells, or dendritic cells. In some embodiments of the methods of the present disclosure, the method may further comprise activating (before or after the expansion step) the isolated cells with a NKT cell activator, a T cell activator, or a dendritic cell activator, This should be detailed above.

In some embodiments, after isolating NKT cells, T cells, or dendritic cells or a population of NKT cells, a population of T cells, or a population of dendritic cells from a subject or a sample derived from a subject, a method protein of the present disclosure It may further comprise the step of introducing a nucleic acid encoding a nucleic acid into the isolated cell or cells. Appropriate methods for introducing nucleic acids into cells are well known to those skilled in the art - including, for example, electroporation, sonication, cell microinjection, microparticle delivery, calcium-phosphate mediated transfection, and liposome based transfection. physical or chemical methods; Alternatively, viral transduction. After introduction of the nucleic acid encoding the protein, the cell or cells may be cultured under conditions that facilitate expression of the encoded protein. Appropriate methods, reagents and conditions for culturing cells are well known to those skilled in the art. A cell (NKT cell, T cell, or dendritic cell) or cells (NKT cell, T cell, or dendritic cell) into which a nucleic acid encoding a protein has been introduced may be referred to herein as a transfected or transformed cell.

In some embodiments of the present disclosure, the nucleic acid encoding the protein is a nucleic acid encoding a protein selected from the group consisting of one or more of: T-cell receptor (TCR), chimeric antibody receptor (CAR), cleavage, and universal and Programmable CAR (SUPRA-CAR).

After isolation, NKT cells, T cells, and/or dendritic cells can be genetically engineered for specific targets. For example, NKT can be expanded by IL-2 and activated with GalCer (galactosylceramide), pulsed self-irradiated PBMC, and then transduced to express CAR or recombinant TCR (rTCR). The CAR or rTCR can specifically bind a target selected from GD2 (disialoganglioside) and CD19. For example, the CAR can be NCT03294954 (binds specifically to GD2) or NCT03774654 (binds specifically to CD19).

Moreover, it may undergo NKT cell, T cell, and/or dendritic target activation. For example, the following procedures may be used: nanovectors for passive and active delivery; a-GalCer-loading APC for targeted activation of NKT on tumors; i.v. of α-GalCer. administration; and/or adding α-GalCer to cultured cells to stimulate bulk PBMCs (2 to 3 times) (to generate an iNKT cell-cell-enriched population and then re-inject into the patient)

Moreover, NKT cells, T cells, and/or dendritic cells may be linked directly to a tumor targeting moiety (tumor cell or TME). Chemical modification of stimulants on NKT cells (polarization of immune response by α-GalCer analogs), T cells, and dendritic cells can also be used.

The term "chimeric antibody receptor" (CAR), as used herein, relates non-exclusively to constructs containing the antigen binding domain of an antibody fused to a potent T-cell activator domain. T-cells modified with the CAR construct can bind antigen and can be stimulated to attack the bound cells. Artificial T cell receptors (also known as chimeric T cell receptors, chimeric immunoreceptors, chimeric antibody receptors (CARs)) are engineered receptors that implant any specificity into immune effector cells. Receptors are called chimeric because they are made up of parts from different sources. Chimeric Antibody Receptor T cells or receptors/ligands or antibodies expressed by cellular immunotherapy may be monospecific or bispecific or multispecific.

In some embodiments, the TCR, CAR, and/or SUPRA-CAR may comprise an antigen binding domain that binds an antigen selected from the group of receptor/ligand/target consisting of: proto-oncogene tyrosine-protein kinase ABL1, TRAIL bound to citrullated antigen, ErbB2/HER2, CD16, WT-1, KRAS, Glypican 3, CD3, CD20, CD226, CD155, CD123, HPV-16 E6, Melan-A/MART-1, DR4 receptor, LMP, MTCR, ESO, NY-ESO-1, gp100, 4SCAR-GD2/CD56, mesodellin (CAK1 antigen or Pre-pre-pro-megakaryocyte enhancing factor or MSLN); DNA synthesis inhibitors; histamine H1 receptor (HRH1) antagonist; Prostaglandin G/H synthase 2 (cyclooxygenase 2 or COX2 or prostaglandin endoperoxide synthetase 2 or PHS II or prostaglandin H2 synthetase 2 or PTGS2 or EC 1.14.99.1) inhibitor, CD19 (B lymphocyte surface antigen B4 or differentiation antigen CD19 or T cell surface antigen Leu 12 or CD19), cell adhesion molecule 5 (carcinoembryonic antigen or CEA or meconium antigen 100 or CD66e or CEACAM5); interleukin 2 receptor (IL2R) agonists, epidermal growth factor receptor (proto-oncogene c ErbB 1 or receptor tyrosine protein kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1); DNA ligase (EC 6.5.1.) inhibitors; DNA ligase (EC 6.5.1.), DNA polymerase alpha (POLA or EC 2.7.7.7) inhibitors; DNA primase (EC 2.7.7.6) inhibitors; ribonucleoside diphosphate reductase (ribonucleotide reductase or RRM or EC 1.17.4.1) inhibitors; RNA polymerase II (RNAP II or Pol II or EC 2.7.7.6) inhibitors, DNA polymerase (EC 2.7.7.7) inhibitors; DNA topoisomerase II (EC 5.99.1.3) inhibitors; CD22, meso, DNA primase (EC 2.7.7.6); programmed cell death 1 ligand 1 (PD L1 or B7 homologue 1 or CD274) inhibitors; RNA polymerase II (RNAP II or Pol II or EC 2.7.7.6), histone lysine N methyltransferase EZH2 (enhancer of ENX 1 or Zeste homolog 2 or lysine N methyltransferase 6 or EZH2 or EC 2.1.1.43) inhibitors ; programmed apoptosis 1 ligand 1 (PD L1 or B7 homologue 1 or CD274), C-X-C chemokine receptor type 4 (FB22 or metasynthesis or HM89 or LCR1 or 7 transmembrane domain receptors from leukocytes or lipopolysaccharide related protein 3 or stromal cells derived factor 1 receptor or NPYRL or CD184 or CXCR4) antagonist; granulocyte colony stimulating factor receptor (CD114 or GCSFR or CSF3R) agonists, adenosine deaminase (adenosine aminohydrolase or ADA or EC 3.5.4.4) inhibitors; Cells against cells expressing tumor necrosis factor receptor superfamily member 17 (B cell maturation antigen or CD269 or TNFRSF17), the inactive tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor related 1 or ROR1 or EC 2.7.10.1) toxicity; T cell surface glycoprotein CD3 epsilon chain (T cell surface antigen T3/Leu 4 epsilon chain or CD3E); dihydrofolate reductase (DHFR or EC 1.5.1.3) inhibitors; ephrin type A receptor 2 (epithelial cell kinase or tyrosine protein kinase receptor ECK or EPHA2 or EC 2.7.10.1) inhibitors; glucocorticoid receptor (GR or nuclear subfamily 3 group C member 1 or NR3C1) agonists; /Stem Cell Growth Factor Receptor Kit (Proto-Oncogene c Kit or Tyrosine Protein Kinase Kit or v Kit Hardy Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog or Pybalt Characteristic Protein or p145 c Kit or CD117 or KIT or EC 2.7.10.1 ) inhibitors; platelet-derived growth factor receptor beta (beta type platelet-derived growth factor receptor or CD140 antigen-like family member B or platelet-derived growth factor receptor 1 or CD140b or PDGFRB or EC 2.7.10.1) inhibitors; tubulin inhibitors; tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Fyn (proto-oncogene Syn or proto-oncogene c Fyn or Src-like kinase or p59 Fyn or FYN or EC 2.7.10.2) inhibitors; tyrosine protein kinase Lck (leukocyte C-terminal Src kinase or protein YT16 or proto-oncogene Lck or T cell specific protein tyrosine kinase or lymphocyte cell specific protein tyrosine kinase or p56 LCK or LCK or EC 2.7.10.2) inhibitors; Tyrosine protein kinase Yes (proto-oncogene c Yes or p61 Yes or YES1 or EC 2.7.10.2) inhibitors, tumor necrosis factor (kakectin or TNF alpha or tumor necrosis factor ligand superfamily member 2 or TNF a or TNF) inhibitors, transcription signal transducer and activator of 3 (acute phase response factor or DNA binding protein APRF or STAT3) inhibitors, Bcr-Abl tyrosine kinase (EC 2.7.10.2) inhibitors; dihydrofolate reductase (DHFR or EC 1.5.1.3); ephrin type A receptor 2 (epithelial cell kinase or tyrosine protein kinase receptor ECK or EPHA2 or EC 2.7.10.1); Mast/Stem Cell Growth Factor Receptor Kit (Proto-oncogene c Kit or Tyrosine Protein Kinase Kit or v Kit Hardy Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog or Pybalt Characteristic Protein or p145 c Kit or CD117 or KIT or EC 2.7. 10.1); platelet derived growth factor receptor beta (beta type platelet derived growth factor receptor or CD140 antigen-like family member B or platelet derived growth factor receptor 1 or CD140b or PDGFRB or EC 2.7.10.1); tubulin; tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Fyn (proto-oncogene Syn or proto-oncogene c Fyn or Src-like kinase or p59 Fyn or FYN or EC 2.7.10.2) inhibitors; tyrosine protein kinase Lck (leukocyte C-terminal Src kinase or protein YT16 or proto-oncogene Lck or T cell specific protein tyrosine kinase or leukocyte cell specific protein tyrosine kinase or p56 LCK or LCK or EC 2.7.10.2) inhibitors; Tyrosine protein kinase Yes (proto-oncogene c Yes or p61 Yes or YES1 or EC 2.7.10.2) inhibitor, caspase 9 (apoptotic protease Mch 6 or apoptosis protease activating factor 3 or ICE-like apoptotic protease 6 or CASP9 or EC 3.4.22.62) activators; Prostate stem cell antigen (PSCA), melanoma antigen preferentially expressed in tumor (cancer/testis antigen 130 or Opa interacting protein 4 or preferentially expressed antigen of OIP4 or melanoma or PRAME), transcription 3 (acute phase response factor) or signal transducer and activator of DNA binding protein APRF or STAT3) inhibitor, CD44 antigen (CDw44 or epican or extracellular matrix receptor III or GP90 leukocyte homing/adhesion receptor or HUTCH I or heparan sulfate proteoglycan or Hermes antigen or hyaluronan nate receptor or phagocytic glycoprotein 1 or CD44), AXL (annexellecto) receptor tyrosine kinase, GAS6, TAM receptor tyrosine kinase, TYRO-3 (also known as Brt, Dtk, Rse, Sky and Tif), AXL (Ark, Also known as Tyro7 and Ufo), and MER (also known as Eyk, Nym and Tyro12), CTLA4, tumor necrosis factor receptor superfamily member 8 (CD30L receptor or Ki 1 antigen or leukocyte activating antigen CD30 or CD30 or TNFRSF8), caspase 9 (apoptotic protease Mch 6 or apoptosis protease activator 3 or ICE-like apoptosis protease 6 or CASP9 or EC 3.4.22.62) activators; cytotoxicity to ganglioside GD2 expressing cells; prostaglandin G/H synthetase 1 (cyclooxygenase 1 or COX1 or prostaglandin endoperoxide synthetase 1 or prostaglandin H2 synthetase 1 or PTGS1 or EC 1.14.99.1) inhibitors; Cytokine, interleukin, claudin 6 (Sculin or CLDN6), NKG2D, MICA, MICB and ULBP 1-6, NKp30, B7H6 (NCR3LG1), Bag6, B7 family, CD40 ligand (T cell antigen Gp39 or TNF related activating protein or tumor necrosis factor ligand superfamily member 5 or CD154 or CD40LG) activators; Interleukin 12 (IL12) activator, Interleukin 3 Receptor Subunit Alpha (IL3RA Obesity/Stem Cell Growth Factor Receptor Kit (Proto-oncogene c Kit or Tyrosine Protein Kinase Kit or v Kit Hardy Zuckerman 4 Feline Sarcoma Viral Oncogene Homolog or Pybalt specific protein or p145 c Kit or CD117 or KIT or EC 2.7.10.1) antagonist; proto-oncogene tyrosine protein kinase receptor Ret (cadherin family member 12 or proto-oncogene c Ret or RET or EC 2.7.10.1) inhibitor ; receptor type tyrosine protein kinase FLT3 (FMS-like tyrosine kinase 3 or FL cytokine receptor or stem cell tyrosine kinase 1 or fetal liver kinase 2 or CD135 or FLT3 or EC 2.7.10.1) antagonist; vascular endothelial growth factor receptor 1 (Fms) tyrosine kinase 1 or tyrosine protein kinase receptor FLT or tyrosine protein kinase FRT or vascular permeability factor receptor or VEGFR1 or FLT1 or EC 2.7.10.1) antagonist;vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insertion domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1) antagonist; vascular endothelial growth factor receptor 3 (Fms-like tyrosine kinase 4 or tyrosine protein kinase receptor FLT4 or VEGFR3 or FLT4 or EC 2.7.10.1) Gil Antibody, caspase 9 (apoptotic protease Mch 6 or apoptosis protease activator 3 or ICE-like apoptosis protease 6 or CASP9 or EC 3.4.22.62) activator, cytotoxic T leukocyte protein 4 (cytotoxic T leukocyte associated antigen 4) or CD152 or CTLA4) antagonist, bone marrow cell surface antigen CD33 (sialic acid binding Ig-like lectin 3 or gp67 or CD33), hepatocytes growth factor receptor (proto-oncogene c Met or tyrosine protein kinase Met or HGF/SF receptor or scatter factor receptor or MET or EC 2.7.10.1), epithelial cell adhesion molecule (adenocarcinoma-associated antigen or cell surface glycoprotein Trop 1 or epithelial cells) surface antigen or epithelial glycoprotein 314 or KS 1/4 antigen or KSA or tumor-associated calcium signal transducer 1 or CD326 or EPCAM), ganglioside GD2, Lewis Y antigen (CD174), latent membrane protein 1 (protein p63 or LMP1) , mucin 1 (breast cancer-associated antigen DF3 or episialin or H23AG or Krebs Von Den Lungen 6 or PEMT or peanut reactive yomucin or polymorphic epithelial mucin or tumor-associated epithelial membrane antigen or tumor-associated mucin or CD227 or MUC1), T cell receptor beta 1 chain C region (TRBC1), vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insertion domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1), BCMA, PD-1 , interleukin-6 receptor, NKR2, CX-072, T leukocyte protein 4 (cytotoxic T leukocyte associated antigen 4 or CD152 or CTLA4) antagonist; Serine/threonine protein kinase B Raf (p94 or proto-oncogene B Raf or v Raf murine sarcoma viral oncogene homologue B1 or BRAF or EC 2.7.11.1) inhibitor, mucin 16 (ovarian cancer-associated tumor marker CA125 or ovarian carcinoma antigen) CA125 or MUC16); Bcr-Abl tyrosine kinase (EC 2.7.10.2) inhibitors; tyrosine protein kinase CSK (C Src kinase or protein tyrosine kinase CYL or CSK or EC 2.7.10.2) inhibitors; tyrosine protein kinase Fyn (proto-oncogene Syn or proto-oncogene c Fyn or Src-like kinase or p59 Fyn or FYN or EC 2.7.10.2) inhibitors; tyrosine protein kinase Lck (leukocyte C-terminal Src kinase or protein YT16 or proto-oncogene Lck or T cell specific protein tyrosine kinase or leukocyte cell specific protein tyrosine kinase or p56 LCK or LCK or EC 2.7.10.2) inhibitors; Tyrosine protein kinase Yes (proto-oncogene c Yes or p61 Yes or YES1 or EC 2.7.10.2) inhibitor, cyclin dependent kinase 1 (p34 protein kinase or cell division protein kinase 1 or cell division control protein 2 homolog or CDK1 or EC 2.7) .11.22 or EC 2.7.11.23) inhibitors; cyclin dependent kinase 2 (p33 protein kinase or cell division protein kinase 2 or CDK2 or EC 2.7.11.22) inhibitors; Granulocyte macrophage colony stimulating factor receptor subunit alpha (CDw116 or CD116 or CSF2RA) agonist, EGFRVIII, tyrosine protein kinase SYK (splenic tyrosine kinase or p72 Syk or SYK or EC 2.7.10.2) inhibitor, alpha fetoprotein (alpha 1 fetoprotein) or alpha fetoglobulin or AFP), cancer/testis antigen 1 (autoimmunogenic cancer/testis antigen or cancer/testis antigen 6.1 or L antigen family member 2 or CTAG1A or CTAG1B); HBV antigen, EGFR family member, herin, tyrosine protein kinase BTK (Bruton's tyrosine kinase or B cell progenitor kinase or agammaglobulinemia tyrosine kinase or BTK or EC 2.7.10.2) inhibitor, CD4, epithelial cell adhesion molecule (adenocarcinoma associated antigen) or cell surface glycoprotein Trop 1 or epithelial cell surface antigen or epithelial glycoprotein 314 or KS 1/4 antigen or KSA or tumor-associated calcium signal transducer 1 or CD326 or EPCAM), prolyl endopeptidase FAP (170 kDa melanoma) Membrane bound gelatinase or dipeptidyl peptidase FAP or integrated membrane serine protease or fibroblast activating protein alpha or gelatinase protease FAP or cepresse or FAP or EC 3.4.21.26 or EC 3.4.14.5), neuronal cell adhesion molecule 1 (antigen recognized by monoclonal antibody 5.1H11 or CD56 or NCAM1); Epidermal growth factor receptor (proto-oncogene c ErbB 1 or receptor tyrosine protein kinase erbB 1 or HER1 or ERBB1 or EGFR or EC 2.7.10.1) antagonist, tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor association 1 or ROR1) or EC 2.7.10.1); Wilms tumor protein (WT33 or WT1); Interleukin 13 receptor subunit alpha 2 (interleukin 13 binding protein or CD213a2 or IL13RA2), feeder glycoprotein (M6P1 or 5T4 tumor-fetal antigen or 5T4 tumor-fetal feeder glycoprotein or Wnt activated inhibitory factor 1 or TPBG), SLAM family member 7 (CD319 or membrane protein FOAP 12 or CD2-like receptor activated cytotoxic cell or Novel Ly9 or protein 19A or CD2 subset 1 or CS1 or SLAMF7), B cell lymphoma 2 (Bcl 2) inhibitor; DNA (cytosine 5) methyltransferase 1 (CXXC type zinc finger protein 9 or DNA methyltransferase HsaI or MCMT or DNMT1 or EC 2.1.1.37) inhibitor, ROR1, CD19&CD40L, avidin (EGFRiiiv), folate receptor, CD30, pmel CD*8 T, CD33, NKR2, epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen, aberrant product of ras, p53, alpha fetal protein (AFP), CA-125, CA15-3, CA27-29, CA19 -9, calcitonin, calretinin, CD34, CD99MIC 2, CD117, chromogranin, cytokeratin (various types: TPA, TPS, Cyfra21-1), desmin, epithelial membrane antigen (EMA), factor VIII, CD31 FL1 , glial fibrotic acid protein (GFAP), cystic disease fluid protein (GCDFP-15), HMB-45, human chorionic gonadotropin (hCG), immunoglobulin, inhibin, keratin (various types), leukocyte markers (various types) type), BCR-ABL, Myo D1, muscle specific actin (MSA), nerve fibers, neuron specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate specific antigen (PSA), PTPRC ( CD45), S100 protein, smooth muscle actin (SMA), synaptophysin, thymidine kinase, thyroglobulin (Tg), thyroid transcription factor-1 (TTF-1), tumor M2-PK, vimentin, SV40, adenovirus E1b -58kd, IGF2B3, ubiquitous (low level), kallikrein 4, KIF20A, lengthin, meloe, MUC5AC, immature laminin receptor, TAG-72, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, EphA3, telomerase, SAP-1, BAGE family, CAGE family, GAGE family, MAGE family, SAGE family, XAGE family, LAGE-1, PRAME, SSX-2, pmel17, Tyrosinase, TRP-1/-2, P. polypeptide, MC1R, β-catenin, prostate specific antigen, BRCA1, BRCA2, CDK4, CML66, fibronectin, MART-2, Ras, TGF-beta receptor II, T cell receptor ( TCR), BLOC1S6, CD10/neprilysin, CD24, CD248, CD5/differentiation cluster 5, CD63/Tspan-30/tetraspanin-30, CEACAM5/CD66e, CT45A3, CTAG1A, CXORF61, DSE, GPA33, HPSE, KLK3 , LCP1, LRIG3, LRRC15, megakaryocyte enhancing factor, MOK, MUC4, NDNL2, OCIAD1, PMPCB, PTOV1, RCAS1/EBAG9, RNF43, ROPN1, RPLP1, SARNP, SBEM/MUCL1, TRP1/TYRP1, CA19-9, inactive tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor association 1 or ROR1 or EC 2.7.10.1), ALK tyrosine kinase receptor (anaplastic lymphoma kinase or CD246 or ALK or EC 2.7.10.1), prostate stem cell antigen (PSCA), Melanoma antigen preferentially expressed in tumor (cancer/testis antigen 130 or Opa interacting protein 4 or OIP4 or preferentially expressed antigen of melanoma or PRAME), transcription 3 (acute phase response factor or DNA binding protein APRF or STAT3) Signal transducers and activators of inhibitors, CD44 antigen (CDw44 or epican or extracellular matrix receptor III or GP90 leukocyte homing/adhesion receptor or HUTCH I or heparan sulfate proteoglycan or Hermes antigen or hyaluronate receptor or phagocytic glycoprotein 1 or CD44), CD40 ligand (T cell antigen Gp39 or TNF associated activating protein or tumor necrosis factor ligand superfamily member 5 or CD154 or CD40LG) activator; tumor necrosis factor receptor superfamily member 13B (transmembrane activator and CAML interactor or CD267 or TACI or TNFRSF13B); Cytotoxic to cells expressing tumor necrosis factor receptor superfamily member 17 (B cell maturation antigen or CD269 or TNFRSF17), CD276 antigen (B7 homolog 3 or 4Ig B7 H3 or costimulatory molecule or CD276), bone marrow cell surface antigen CD33 (sialic acid binding Ig-like lectin 3 or gp67 or CD33), ADP ribosyl cyclase/cyclic ADP ribose hydrolase 1 (cyclic ADP ribose hydrolase 1 or T10 or 2' phospho ADP ribosyl cyclase) Second/2' phospho cyclic ADP ribose transferase or ADP ribosyl cyclase 1 or CD38 or EC 3.2.2.6 or EC 2.4.99.20), type C lectin domain family 14 member A (epidermal growth factor receptor 5 or EGFR5) or CLEC14A), hepatocyte growth factor receptor (proto-oncogene c Met or tyrosine protein kinase Met or HGF/SF receptor or scatter factor receptor or MET or EC 2.7.10.1), epithelial cell adhesion molecule (adenocarcinoma associated antigen or cell surface glycoprotein) Trop 1 or epithelial cell surface antigen or epithelial glycoprotein 314 or KS 1/4 antigen or KSA or tumor-associated calcium signal transducer 1 or CD326 or EPCAM), ganglioside GD3, interleukin 13 receptor subunit alpha 2 (interleukin 13 binding protein) or CD213a2 or IL13RA2); kappa myeloma antigen (KMA), lambda myeloma antigen (LMA), latent membrane protein 1 (protein p63 or LMP1), melanoma-associated antigen, T leukocyte activating antigen CD80 (activating B7-1 antigen or CTLA 4 counter receptor B7.1 or cytotoxicity to cells expressing CD80); Cytotoxic, inactive tyrosine protein kinase transmembrane receptor ROR1 (neurotrophic tyrosine kinase receptor association 1 or ROR1 or EC 2.7.10.1), Fas apoptosis inhibitory molecule 3 (IgM Fc fragment receptor or modulator of Fas-induced apoptosis Toso or TOSO or FAIM3 or FCMR), T cell receptor beta 1 chain C region (TRBC1), vascular endothelial growth factor receptor 2 (fetal liver kinase 1 or kinase insertion domain receptor or protein tyrosine kinase receptor flk 1 or VEGFR2 or CD309 or KDR or EC 2.7.10.1), alpha fetoprotein (alpha 1 fetoprotein or alpha aglobulin or AFP), cancer /testis antigen 1 (autoimmunogenic cancer/testis antigen NY ESO 1 or cancer/testis antigen 6.1 or L antigen family member 2 or CTAG1A or CTAG1B), T cell surface glycoprotein CD5 (leukocyte antigen T1/Leu 1 or CD5), prolyl endopeptidase FAP (170 kDa melanoma membrane bound gelatinase or dipeptidyl peptidase FAP or integrated membrane serine protease or fibroblast activating protein alpha or gelatinase protease FAP or seprase or FAP or EC 3.4. 21.26 or EC 3.4.14.5), neuronal cell adhesion molecule 1 (antigen recognized by monoclonal antibody 5.1H11 or CD56 or NCAM1), type C lectin domain family 12 member A (myeloid inhibitory type C lectin-like receptor or dendritic cell-associated) lectin 2 or type C lectin-like molecule 1 or CLEC12A), integrin alpha V (vitronectin receptor subunit alpha or CD51 or ITGAV); Cytotoxic to cells expressing integrin beta 6 (ITGB6), interleukin 13 receptor subunit alpha 2 (interleukin 13 binding protein or CD213a2 or IL13RA2), feeder glycoprotein (M6P1 or 5T4 tumor fetal antigen or 5T4 tumor fetal feeder cells) Glycoprotein or Wnt activated inhibitory factor 1 or TPBG), feeder glycoprotein (M6P1 or 5T4 oncofetal antigen or 5T4 oncofetal feeder glycoprotein or Wnt activated inhibitory factor 1 or TPBG), type C lectin domain family 12 member A (myeloid inhibitory type C lectin-like receptor or dendritic cell-associated lectin 2 or type C lectin-like molecule 1 or CLEC12A), SLAM family member 7 (CD319 or membrane protein FOAP 12 or CD2-like receptor activated cytotoxic cell or Novel Ly9 or protein 19A or CD2 subset 1 or CS1 or SLAMF7), SLAM family member 7 (CD319 or membrane protein FOAP 12 or CD2-like receptor activated cytotoxic cells or Novel Ly9 or protein 19A or CD2 subset 1 or CS1 or SLAMF7), immune globulin, multidrug resistance-associated protein 3 (MRP3), proto-oncogene tyrosine-protein kinase ABL1, prostatic acid phosphatase, OY-TES-1, ACSM2A, alpha-actinin-4, perilipin-2, alpha-fetoprotein, Lymphoblastic crisis oncogene (Lbc) oncoprotein, aldehyde dehydrogenase 1 family member A1 (ALDH1A1), AML, ANKRD17, NY-BR-1, Annexin II, ARHGAP17, ARHGAP30, ARID1B, endoplasmic reticulum resident protein, 5' -Aminoimidazole-4-carboxamide-1-beta-d-ribonucleotide transformylase/inosinicase (AICRT/I), ATR, ATXN2, ATXN2L, BAGE1, BCL11A, Bcl-xL, breakpoint cluster realm, survivin, livin/ML-IAP, HM1.24, BTB domain containing 2 (BTBD2), C6ORF89, carbonic anhydrase IX, CLCA2, CRT2, CAMEL, CAN protein, caspase-5, caspase-8, KM-HN-1, CCDC88B, cyclin B1, cyclin D1, CCNI, CDC2, CDC25A, CDC27, CDK12, intestinal carboxylesterase, CEP95, CHAF1A, coactosin-like 1, CPSF, CRYBA1, TRAG-3, macrophage colony stimulating factor, CSNK1A1, melanoma-associated chondroitin sulfate proteoglycan (MCSP) , cathepsin H, other cued lung cancer antigen 1, P450 1B1 or CYP1B1, DDR1, DEK oncogene, DEK-CAN, Dickkopf-1 (DKK1), DNAJC8, DSCAML1, EEF2, elongation factor containing Tu GTP binding domain or SNRP116, EIF4EBP1 , human mena protein, EP300, ETV5, TEL1 or ETV6, polycomb population protein enhancer of zeste homolog 2 (EZH2), F2R, F4.2, FAM53C, fibroblasts, growth factor 5 or FGF5, formin involvement of leukocyte 1 Protein (FMNL1), Fibromodulin (FMOD), FNDC3B, FKHR, GDP-L-fucose, GAS7, GFI1, GIGYF2, GPNMB, O, A1, GPSM3, GRK2, GRM5, H3F3A, HAUS3, HERC1, HERV- K-MEL, HIVEP2, HMGN, HMHA1, heme oxygenase-1 (HO-1), HNRPL, heparanase, HMSD-v-encoding mHA, HSPA1A, Hsp70, HSPB1, immediate early response gene X-1 (IEX -1), insulin-like growth factor (IGF)-II mRNA binding protein 3 (IMP-3), IP6K1, IRS2, ITGB8, JUP, RU2AS, KANSL3, KLF10, KLF2, KLK4, KMT2A, K-ras, low density lipid receptor (LDLR), LDLR-FUT, Mac-2-binding protein, KIAA0205, LPP, LR P1, LRRC41, LSP1, LUZP1, leukocyte antigen 6 complex locus K (LY6K), MACF1, MAP1A, MAP3K11, MAP7D1, matrilin-2, Mcl-1, MDM2, malate, MEF2D, MEFV, milk bladder membrane protein BA46 ( lactaderin), melanotransferrin, GNT-V or N-acetylglucosaminintransferase V, MIIP, MMP14, matrix metalloproteinase-2, MORC2, melanoma antigen p15, MUC2, MUM, MYC, MYL9, non classical myosin class I genes, N4BP2, NCBP3, NCOA1, NCOR2, NFATC2, NFYC, NIFK, ninein, NPM, NPM1-ALK1, N-ras, OAS3, P polypeptide, OGT, OS-9, ErbB3-binding protein 1, PAGE-4, P21-activated serine kinase 2 (PAK2), neo-PAP, PARP12, PAX3, PAX3-FKHR, PCBP2, phosphoglycerate kinase 1 (PKG1), PLEKHM2, promyelocytic leukemia or PML, PML- RARA, POLR2A, cyclophilin B, PPP1CA, PPP1R3B, peroxyredoxin 5, proteinase 3, parathyroid hormone related protein (PTHrP), receptor-like protein tyrosine phosphatase kappa, MG50, NY-MEL-1 or RAB38, RAGE , RALGAPB, RAR alpha, RBM, RCSD1, Recoverin, RERE, RGS5, RHAMM/CD168, RPA1, ribosomal protein L10a, ribosomal protein S2, RREB1, RSRP1, RTCB, SART, SCAP, mammaglobin A, secernin 1, SDCBP , SETD2, SF3B1, renal ubiquitous protein 1, SIK1, SIRT2, SKI, hairpin binding protein, SLC35A4, prosteine, SLC46A1, SNRPD1, SOGA1, SON, SOX10, SOX11, SOX2, SOX-4, sperm protein 17, SPEN, SRRM2 , SRSF7, SRSF8, SSX1, SSX2 or HOM -MEL-40, SSX4, STAT1, STEAP, STRAP, ART-1, SVIL, HOM-TES-14/SCP1, CD138, SYNM, SYNPO, SYT, SYT15, SYT-SSX1, SYT-SSX2, SZT2, TAPBP, TBC1D10C , TBC1D9B, hTERT, THNSL2, THOC6, TLK1, TNS3, TOP2A, TOP2B, ATP-dependent interferon-reactivity (ADIR), TP53, triose phosphate isomerase TPI1, tropomyosin-4, TPX2, TRG, T-cell receptor Gamma Alternate Reading Frame Protein (TARP), TRIM68, prostate-specific protein transient receptor potential-p8 (trp-p8), TSC22D4, TTK protein kinase (TTK), thymidylate synthetase (TYMS), UBE2A, ubiquitin conjugation enzyme variants Kua, COA-1, USB1, NA88-A, VPS13D, BING4, WHSC1L1, WHSC2, WNK2, WT1, XBP1, XPO1, ZC3H14, ZNF106, ZNF219, papillomavirus binding factor (PBF), E3 ubiquitin-protein ligase UBR4 .

In some embodiments of the present disclosure, the TCR, CAR, and/or SUPRA-CAR may not comprise an antigen-binding domain that binds an antigen selected from the above recited group of receptor/ligand/target.

In some preferred embodiments, the TCR, CAR, and/or SUPRA-CAR may comprise an antigen-binding domain that binds to an antigen selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3 , CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, Her2.

Following introduction of the nucleic acid encoding the protein, NKT cells, T cells, or dendritic cells or NKT cells, T cells, or dendritic cells can be expanded in culture. Methods and reagents suitable for cell culture and expansion are well known to those skilled in the art. After expansion, the methods of the present disclosure may further comprise activating the cells with an NKT cell activator, a T cell activator, or a dendritic cell activator. NKT Cell Activators T cell activators, or dendritic cell activators, may be described in detail above.

In some embodiments, the AVM_NKT, AVM-T cell, and/or AVM-dendritic cell or targeted AVM_NKT, AVM-T cell, and/or AVM-dendritic cell is a nucleic acid, dsRNA, siRNA, micro RNA, dsDNA, ssDNA, cDNA, rRNA, mRNA, tRNA, siRNA, dsRNAi, RNAi, organic compound, cytotoxic drug, antibody, vedotin, ozogamicin, emtansine, deruxtecan, mertansine, mapodotin, tubulin inhibitor, mono Monomethyl auristatin-F (MMAF), a peptide analog of methyl auristatin-E (MMAE) and dolastatin-10, maytansinoid, vinca alkaloid, calicheamicin, duocarmycin, pyrrolobenzodiazepine dimer , thaliline, tesirin, indolinobenzodiazepine pseudodimer, sorabtansine, DM1, DM4, neurotransmitter, DNA intercalator, antimetabolite, endostatin, neurotrophin, chemotherapy, or growth factor, or antibody, toxin , radioactive, antibiotic, antifungal, anti-viral, receptor, virus, cytokine, lipid, chemokine, peptide and protein, anti-parasitic agent, hormone, antigen, neuroactive agent, receptor agonist or antagonist, small molecule, or any type used to deliver payloads such as biological payloads or biologically active payloads.

In some embodiments of the present disclosure, cells of the present disclosure can be used to deliver a payload that is not one or more of the payloads recited above.

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The disclosure also provides a method of treating cancer, an autoimmune disease, or an infectious disease (also referred to as a microbial disease) in a subject. In some embodiments, the method of treatment is a method of generating a population of natural killer T cells (NKT cells) in a subject as specifically described. In some embodiments, the method of treatment is a method of recruiting a population of NKT cells in a subject as described elsewhere herein. In some embodiments, the method of treatment is a method of generating a T cell population in a subject as detailed above. In some embodiments, the method of treatment is a method of generating a population of dendritic cells in a subject as detailed above. In some embodiments, the method of treatment is a method of generating a population of NKT cells, T cells, and/or dendritic cells in a subject as detailed above. In another embodiment, the method of treating is a method comprising administering to a subject a therapeutically effective dose of isolated NKT cells, T cells, and/or dendritic cells of the present disclosure. These are any of the isolated NKT cells or populations of NKT cells, isolated T cells or populations of T cells, and isolated dendritic cells or populations of dendritic cells summarized above, the expanded and non-expanded, and/or activation described above. inactivated or non-activated and/or transfected or non-transfected cells. In these embodiments, the subject, cancer, autoimmune disease, infectious disease, and/or mechanism of therapeutic efficacy may be as detailed above.

In embodiments where the method of treatment comprises administering to the subject a therapeutically effective dose of isolated NKT cells, T cells, and/or dendritic cells of the present disclosure, the subject to which the isolated cells are administered may be the same as the subject. In such embodiments, the treatment may be referred to as autologous cell therapy. The term "autologous" refers to any substance derived from the same subject, whether the subject is a human or another animal, which is later reintroduced. In another embodiment, wherein the method of treatment comprises administering to the subject a therapeutically effective dose of isolated NKT cells, T cells, and/or dendritic cells of the present disclosure, the subject to which the isolated cells are administered different from the isolated subject. In such embodiments, the treatment may be referred to as allogeneic cell therapy. The term “allogeneic” refers to the introduction of any substance derived from an individual, whether human or other animal, into another individual of the same species. That is, in embodiments wherein the method of treatment comprises administering to a subject a therapeutically effective dose of isolated NKT cells, T cells, and/or dendritic cells of the present disclosure, the cells are from an autologous or allogeneic source. can

A method of treating cancer, an autoimmune disease, or an infectious disease in a subject according to the present disclosure may further comprise administering to the subject a NKT cell activator, a T cell activator, and/or a dendritic cell activator. have. These may be as detailed above.

As used herein, the term “administering” refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as injection or infusion. The phrase “parenteral administration” as used herein means administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, lesion. Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intertracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injections and infusions, as well as in vivo electroporation including perforation. In some embodiments, the formulations disclosed herein can be administered by a non-oral route, eg, orally. Other parenteral routes include topical, epidermal, or mucosal routes of administration, eg, intranasal, intravaginal, rectal, sublingual or topical administration.

The phrase “systemic injection” as used herein refers to, inter alia, intravenous, intraperitoneal, subcutaneous, subnasal, lingual, via bronchoscopy, intravenous, intraarterial, intramuscular, intraocular, intrastriatal, subcutaneous , intradermally, by dermal patches, by skin patches, by patches, into cerebrospinal fluid, into portal vein, into brain, lymphatic system, intrapleurally, retroorbitally, intradermally, intraspleen, intralymphatic.

The term 'injection site' as used herein is particularly intended for intratumoral, or intramuscular, intraocular, intrastriatal, intradermal, intradermal patches such as kidney or liver or pancreas or heart or lung or brain or spleen or eye. , by skin patches, by patches, by cerebrospinal fluid, by the brain.

In some preferred embodiments of the present disclosure, the glucocorticoid receptor modulator may be administered orally. In embodiments wherein the method of treatment of the present disclosure is a method comprising administering to a subject a therapeutically effective dose of isolated NKT cells, T cells, and/or dendritic cells of the present disclosure, the cell is a collagen matrix, extracellular Compositions such as matrix compositions, biopolymer microfilaments made of fibrin or other extracellular matrix materials, patches containing extracellular matrix and biodegradable materials, fibrin patches, alginate or agarose-based patches, extracellular matrix materials and dextran Scaffolds composed of biodegradable, physiologically inactive substances that may be non-exclusively related to components, coating stem cells with organ-specific antigens or binding molecules, scaffolds from ex vivo digested organ donors or cadaveric organs or known as decellularized organs It can be applied directly to the organ or tumor through the residual extracellular matrix, and contact lenses. Preferably the cells are administered to the subject by a method selected from the group consisting of: intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, intracerebrospinal fluid (CSF) injection, intratumoral direct injection, or solid Gel on or near the tumor.

In some embodiments of the present disclosure, the route of administration for the agents and cells disclosed herein may not be one or more of the routes recited above.

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The present disclosure also provides a method of producing a population of natural killer T cells (NKT cells), a method of producing a population of T cells, and/or a method of activating a population of dendritic cells, as detailed above. Corticoid receptor (GR) modulators and ICAM3 modulators are provided. The present disclosure also provides a glucocorticoid receptor (GR) modulator and an ICAM3 modulator for use in a method of treating cancer, an autoimmune disease, or an infectious disease (also referred to as a microbial disease) in a subject as detailed above, wherein A method of treatment is a method of producing/activating/mobilizing a population of natural killer T cells (NKT cells) in a subject. A preferred embodiment is a method for producing a population of natural killer T cells (NKT cells), a method for producing a population of T cells, and/or a method for activating a population of dendritic cells as detailed above. corticoids, and glucocorticoids for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, wherein the method of treatment comprises a population of natural killer T cells (NKT cells) in a subject as detailed above a method of producing a , a method of producing a population of T cells, and/or a method of activating a population of dendritic cells. Another preferred embodiment comprises a glucocorticoid for use in a method of recruiting a population of NKT cells as detailed above. In some particularly preferred embodiments, the glucocorticoid is dexamethasone.

The present disclosure also provides for use in a method of generating a population of natural killer T cells (NKT cells), a method of generating a population of T cells, and/or a method of activating a population of dendritic cells as detailed above. Provided is the use of a glucocorticoid receptor (GR) modulator or an ICAM3 modulator in the manufacture of a medicament for The present disclosure also provides the use of a glucocorticoid receptor (GR) modulator or ICAM3 modulator in the manufacture of a medicament for use in a method of treating cancer, an autoimmune disease, or an infectious disease (also referred to as a microbial disease) in a subject, wherein the method of treatment is a method of producing a population of natural killer T cells (NKT cells) in a subject, a method of producing a population of T cells, and/or a method of activating a population of dendritic cells as detailed above.

The present disclosure also provides the use of a glucocorticoid receptor (GR) modulator or ICAM3 modulator to induce a population of natural killer T cells (NKT cells), wherein the population of natural NKT cells is in a subject as detailed above. induced by a method that produces a population of natural killer T cells (NKT cells). The present disclosure also provides the use of a glucocorticoid receptor (GR) modulator or ICAM3 modulator to induce a population of T cells, wherein the population of T cells produces a population of T cells in a subject as detailed above. induced by the method. The present disclosure also provides the use of a glucocorticoid receptor (GR) modulator or ICAM3 modulator for activating a population of dendritic cells, wherein the population of dendritic cells activates a population of dendritic cells in a subject as detailed above. activated by the method.

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The disclosure also provides a method of producing an induced pluripotent stem cell (iPSC), the method comprising reprogramming an NKT cell, T cell, or dendritic cell of the disclosure to produce an iPSC. The NKT cells, T cells, or dendritic cells of the present disclosure used in the method for producing iPSCs are methods of producing a population of natural killer T cells (NKT cells), T cells, or dendritic cells in a subject as detailed above. NKT cells produced and isolated by

In some embodiments of the disclosed methods of producing iPSCs, reprogramming comprises introducing into a cell of the present disclosure one or more expression cassettes encoding Oct3/4, Klf4, Sox2, and C-myc. In some embodiments, reprogramming comprises introducing Oct3/4, KLF4, Sox2, and c-myc encoding mRNAs into the cell. In some other embodiments of the disclosed methods of producing iPSCs, reprogramming may further comprise introducing into the cell one or more expression cassettes encoding one or more of the following: Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28. In other embodiments, reprogramming may further comprise introducing one or more of the following into the cell: Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28 encoding mRNA. Suitable methods for introducing an expression cassette or encoding mRNA into cells are well known to those skilled in the art - for example electroporation, cell microinjection, or liposome based transfection methods. Reprogramming of non-pluripotent cells in iPSCs using retroviral systems, including lentiviral and adenoviral systems, has been described (Stadtfeld et al, 2008, incorporated herein by reference in its entirety). Reprogramming of adult cells into iPSCs can also be accomplished via plasmids without the use of viral transfection systems (Okita et al, 2008, incorporated herein by reference in its entirety).

Oct-3/4

Oct-3/4 (Pou5f1; cDNA available from Bioclone, San Diego CA) is one of the octameric (“Oct”) transcription factors family and plays an important role in maintaining pluripotency. Induces spontaneous feeder differentiation of Oct-3/4 in Oct-3/4+ cells such as blastomeres and embryonic stem cells, and thus the presence of Oct-3/4 gives rise to the pluripotency and differentiation potential of embryonic stem cells . Various other genes of the "Oct" family, including Oct3/4's close relatives, Oct1 and Oct6, do not induce induction, thus demonstrating the exclusiveness of Oct-3/4 in the induction process.

Klf Family:

Klf4 of the Klf family of genes is a factor for the generation of mouse iPS cells. Klf2 (cDNA available from Bioclone, Inc., San Diego, CA) and Klf4 (cDNA available from Bioclone, Inc., San Diego, CA) are factors capable of generating iPS cells, and the related gene Klf1 (Bioclone , Inc., cDNA available from San Diego, CA) and Klf5 (cDNA available from Bioclone, Inc., San Diego, CA) also had reduced efficiency but also did.

Sox family

The Sox family of genes is associated with maintaining pluripotency similar to Oct-3/4, but is associated with pluripotent and unipotent stem cells, unlike Oct-3/4, which is only expressed in pluripotent stem cells (Bowles et al, 2000). , which is incorporated herein by reference in its entirety). Although Sox2 (cDNA available from Bioclone, San Diego, CA) was the initial gene used for induction, other genes in the Sox family were also found to function in the induction process. Sox1 (cDNA available from Bioclone, Inc., San Diego, CA) generates iPS cells with similar efficiency to Sox2, and the genes Sox3 (human cDNA available from Bioclone, Inc., San Diego, CA), Sox15, and Sox18 is also less efficient, but produces iPS cells.

Myc family

The Myc family of genes are proto-oncogenes associated with cancer. C-myc (cDNA available from Bioclone, Inc., San Diego, CA) is a factor implicated in the generation of mouse iPS cells. However, c-myc may not be required for the generation of human iPS cells. The use of the "myc" gene family in the derivation of iPS cells is problematic for the ultimate potential of iPS cells as a clinical therapy, as 25% of mice transplanted with c-myc-derived iPS cells developed lethal teratoma. have. N-myc (cDNA available from Bioclone, Inc., San Diego, CA) and L-myc were found to induce instead of c-myc with similar efficiency.

Nanog

In embryonic stem cells, Nanog (cDNA available from Bioclone, Inc., San Diego, CA) is required to promote pluripotency along with Oct-3/4 and Sox2 (Chambers et al, 2003, herein in its entirety) incorporated by reference).

LIN28

LIN28 (cDNA available from Bioclone, Inc., San Diego, CA) is an mRNA binding protein expressed in embryonic stem cells and embryonic carcinoma cells involved in differentiation and proliferation (Moss & Tang, 2003, incorporated herein by reference in its entirety). included).

In some embodiments, the disclosed method of producing an iPSC further comprises inducing differentiation of the iPSC of the present disclosure. In some preferred embodiments, the disclosed methods may further comprise inducing differentiation of iPSCs of the present disclosure into NKT cells. Accordingly, the disclosure also provides a method for generating a population of NKT cells, said method comprising differentiating iPSCs produced by a method according to the present disclosure into an NKT lineage. In some embodiments, the disclosed methods may further comprise inducing differentiation of iPSCs of the present disclosure into T cells. Accordingly, the present disclosure also provides a method for producing a population of T cells, said method comprising differentiating iPSCs produced by a method according to the present disclosure into a T cell lineage. In other embodiments, the disclosed methods may further comprise inducing differentiation of iPSCs of the present disclosure into dendritic cells. Accordingly, the present disclosure also provides a method for producing a population of dendritic cells, said method comprising differentiating iPSCs produced by a method according to the present disclosure into a dendritic cell lineage. Such differentiated cells can be used in a method of treating cancer, an autoimmune disease, or an infectious disease (also referred to as a microbial disease) in a subject according to the present disclosure.

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The present disclosure also provides isolated NKT cells, isolated T cells, and isolated dendritic cells produced or recruited by any method disclosed herein, as well as NKT cells produced or recruited by any method disclosed herein; T cells, and an isolated population of dendritic cells are provided. Also in the isolated populations of NKT cells, T cells, and dendritic cells, and NKT cells, T cells, and dendritic cells characterized by a pattern of surface proteins detailed elsewhere herein, and in the methods of treatment of the present disclosure Uses of such cells are provided.

Example

The following examples show that, in addition to inducing near complete lymphocytic depletion of peripheral blood lymphocytes (without affecting cell numbers of neutrophils, platelets, RBCs and stem cells), high dose glucocorticoid receptor agonists, novel populations of NKT cells and T In addition to inducing the production of cells, we demonstrate that it is possible to recruit new populations of activated dendritic cells.

In addition to presenting the known properties of NKT cells, these examples also show that NKT cell populations induced by high-dose glucocorticoid receptor agonists have novel expression patterns of surface proteins, which allow them to directly engulf cancer cells and to treat solid cancers. Demonstrates enhanced cytotoxic efficacy.

Thus, high-dose glucocorticoid agonists represent a promising therapy for use in the treatment of diseases mediated by immune cells such as cancer and lymphocytes.

Materials and Methods

Acute high dose dexamethasone may also be referred to herein as Dex, AugmenStem™, PlenaStem™ or AVM0703. The new population of NKT cells induced after administration of acute high dose dexamethasone (AVM0703) may also be referred to herein as AVM-NKT cells. The new population of T cells induced after administration of acute high dose dexamethasone (AVM0703) may also be referred to herein as AVM-T cells. The new population of dendritic cells induced after administration of acute high dose dexamethasone (AVM0703) may also be referred to herein as AVM-dendritic cells.

For the initial lymphatic depletion study, naive C57Bl/6 mice were treated with 18 mg/kg HED DP by oral gavage. Male C57BL/6 mice were obtained from Taconic Bioscience (Germantown, NY) and acclimatized to laboratory conditions for at least 1 week. Mice were dosed orally once with 18 mg/kg dexamethasone phosphate (DP) or placebo and maintained until a specific time point. Each dosing group was accompanied by a placebo group of the same age and condition according to Table 3. GLP grades AVM0703 and placebo were administered at time points 24 hours, 48 hours, 72 hours, 5 days, 7 days, 11 days, 13 days. Dosing was performed using GMP grade AVM0703 and placebo at time points 6 hours, 21 days, 28 days, and 35 days. When mice reached the study time point, they were euthanized as follows. Mice were anesthetized with isoflurane gas. Once anesthetized, blood is drawn via cardiac puncture and immediately placed in a microtube containing heparin. 10 mL of 5 U/mL of heparin/PBS was injected by slow push for retrograde perfusion through the abdominal aorta to clear any remaining blood from the vasculature. Subsequently, 250 uL of blood was transferred into lavender-lined EDTA-lined microtubes and carried by Lynette Brown of Flow Contract Site Labs (Bothell, WA) for analysis by flow cytometry. The remaining blood was sent to Phoenix Labs (Mukilteo, WA) for complete blood counts and clinical chemistry.

For characterization of the population of induced NKT cells (AVM-NKT), naive C57Bl/6 mice were treated with high dose AVM0703 at 12-45 mg/kg HED DP by oral gavage. Peripheral blood was subsequently examined by flow cytometry at predetermined time intervals to characterize different immune populations. Two NKT populations were identified after treatment with AVM0703: NKT cells defined as CD3medCD49b+ and a novel AVM-NKT population defined as CD3highCD49b+. AVM-NKT cells can be isolated using fluorescence-activated cell sorting (FACS) for CD3highCD49b+ (CD3 high = 2 x 10 ⁴ or greater mean fluorescence intensity).

Example 1 - Acute High Dose of Glucocorticoid Receptor Agonists Cause Near Complete Lymph Depletion of Peripheral Blood Lymphocytes, But Induce a Unique Population of NKT Cells

In the international patent application PCT/US2019/054395, the present authors present a series of demonstrations demonstrating that high-dose glucocorticoid receptor agonists can not only cause near complete lymphocytic depletion of peripheral blood lymphocytes, but also reduce the number of germinal centers of lymphoid organs and deplete thymic lymphocytes. presented the experiment. This effect is achieved without substantially affecting the cell number of neutrophils, platelets, RBCs and stem cells (both hematopoietic stem cells, HSCs, and mesenchymal stem cells, MSCs).

Here, a study conducted in naive mice showed that administration of high doses of glucocorticoid receptor agonists did not substantially affect the cell counts of neutrophils, platelets, red blood cells (RBCs) and stem cells (both HSCs and MSCs), while It has been shown to induce near complete lymphatic depletion. Interestingly, high-dose glucocorticoid receptor agonists have also been shown to induce upregulation of NKT cells.

As shown in Figure 1, high-dose dexamethasone (18 mg/kg HED DP) significantly reduced absolute lymphocyte counts (ALC-NK and NKT cells) compared to placebo - an effect lasting up to 21 days after administration. Near complete lymph node dissection was observed at 6 and 48 hours post-dose, an effect similar to that achieved with standard Cy/Flu chemotherapy (13 mg/kg HED cyclophosphamide and 0.8 mg/kg HED fludarabine).

High-dose dexamethasone selectively eliminates T and B lymphocytes (equivalent to standard Cy/Flu chemotherapy; FIG. 2), monocytes (better than Cy/Flu chemotherapy; FIG. 3) and lymphocyte depletion of neutrophils at target clinical doses ( Fig. 4). Basophils (decreased only at 6 h time points), eosinophils (decreased only at 24 and 48 h time points), platelets (see Fig. 5), and RBCs were all conserved, whereas HSCs (Fig. 6) and MSCs were either conserved or increased. ( ^* p <0.05;^#p< 0.0001).

Surprisingly, it was shown that high dose dexamethasone also induces NKT upregulation ( FIG. 7 ) and generation of a novel NKT cell population (AVM-NKT). These novel AVM-NKT cells are CD49b+ and CD3 very bright (CD3highCD49b+) when examined by flow cytometry. The previously described NKT cells express CD3 with a 1 log lower MFI than AVM-NKT cells (CD3medCD49b+; FIG. 8). AVM-NKT cells appear in the blood of mice 48 hours after administration of high doses (HED 18.1 mg/kg) of the glucocorticoid receptor agonists dexamethasone and betamethasone, but are not induced by standard Cy/Flu chemotherapy.

Dose escalation studies have shown that a single dose between 6-12 mg/kg HED dexamethasone base can induce AVM-NKT cells. 15 mg/kg HED dexamethasone base induces particularly potent production of AVM-NKT cells, as with the 6+6 mg/kg HED dosing schedule.

Example 2 - AVM-NKT Cells Responsible for T and B Lymph Node Resection In Vivo

Mononuclear cells obtained from the peripheral blood of naive male C57Bl/6 mice or single cell splenocytes were incubated with AVM0703 at the same concentration achieved by the highest blood concentration of acute high dose AVM0703 in vivo. No cell death was observed until 72 hours after AVM0703 was added to peripheral blood mononuclear cells or single cell splenocytes in vitro. The lack of in vitro apoptosis in peripheral blood mononuclear cells or splenocytes indicates that lymph node dissection in vivo is primarily due to induction of AVM-NKT cells.

Example 3 - AVM-NKT cells home to tumor sites

In a preliminary study, naïve C57Bl/6 mice were treated with high-dose dexamethasone along with flow cytometrically tested peripheral blood at predetermined time intervals to characterize different immune populations. After treatment with high-dose dexamethasone, two NKT populations were identified: NKT cells defined as CD3medCD49b+ and a novel population of AVM-NKT defined as CD3highCD49b+ ( FIG. 8 ).

AVM-NKT cells were found to appear in the blood of naive mice 48 hours after dexamethasone (AVM0703) or betamethasone at a superpharmaceutical dose (HED 18.1 mg/kg). Conversely, these cells are not induced by standard Cy/Flu chemotherapy or to a significant extent by methylprednisone.

As shown in Figure 9 and Table 2, in normal mice, AVM-NKT cells were induced in the spleen within 48 hours after dexamethasone administration, and clearly appeared in the peripheral blood from 48 hours after dexamethasone administration, and clearly in the bloodstream until 13 days after dexamethasone administration. maintain. AVM-NKT cells were not detected in the spleen of naive placebo-treated mice. Cyclophosphamide/fludarabine administration does not induce this novel NKT population.

Table 2: Presence of AVM-NKT cells in blood, spleen and tumors of naive and A20 mice with and without AVM0703 treatment

In contrast to the time course of AVM-NKT upregulation observed in disease-free normal mice, quantification of AVM-NKT cells in A20 B-cell lymphoma tumor bearing mice found that AVM-NKT cells were absent in peripheral blood. Instead, AVM-NKT cells in these tumor-bearing mice were shown to be home to the tumor site - a site where increased necrosis was evident when irradiated 48 hours after dexamethasone administration ( FIG. 10 ).

Consistent with this, high-dose dexamethasone was shown to significantly retard tumor growth in the A20 model ( FIG. 11 ; Example 15). As A20 cells undergo only about 30% apoptosis for 72 hours after high-dose dexamethasone treatment in vitro, it is believed that AVM-NKT cells have a role in regulating tumor growth.

At harvest, 2 million A20 B lymphoma cells at a density of 1.8e7 cells/mL were mixed with an equal volume of Metrigel (100 ul each) and injected subcutaneously into the left flank of BALB/c mice (200 ul total volume), B A solid tumor model of cell lymphoma was generated. After tumors were established (approximately 7 days or about ˜100-150 mm ³ , which are well-established tumors), mice were treated according to the dosing table shown below. Tumor volume was measured with a caliper three times a week and tumor volume was calculated using the equation V=L x W2 x 0.5. Body weights were also measured 3 times a week and on dosing days to determine the appropriate dose. Mice were considered the study endpoint once tumor volume reached 1500 mm ³ or body weight loss was greater than 20%. When the mice reached the study endpoint, they were euthanized as follows. Mice were anesthetized with isoflurane gas. Once anesthetized, blood was drawn via cardiac puncture and then perfused with 10 mL of 5 U/mL heparin/PBS. The tumor was removed from the right flank by peeling the right back of the mouse. The skin was stretched and fixed and the tumor was isolated from the skin by gently scraping with a scalpel. After the tumor was fixed for 48 hours, it was transferred to 70% ethanol and stored in a cassette at 4°C. Tumors were shipped to HistotoxLabs (Bolder, CO) for excision and staining. Tumor NKT cells were identified by NKp46 staining.

Example 4 - Hematological Cancer Enhances the Concentration of AVM-NKT Cells in Peripheral Blood.

Mice were inoculated with T or B cell lymphomas by tail vein injection of 1-5M lymphoma cells in log growth phase. After 6 hours to 13 days, blood is drawn from the mice and the number of AVM-NKT in the blood is measured by flow cytometric gating for CD3 very high (MFI at least 0.5 higher than T lymphocytes) and CD49b positive cells or by gating for NKp46. . Compared to mice bearing naive or solid tumors, such as T or B lymphoma cells encased in Matrigel and implanted subcutaneously in the flanks, mice with circulating T or B lymphoma cells had significantly increased AVM-NKT counts in the peripheral blood.

Example 5 -AVM-NKT is induced in bone marrow and adipose tissue 48 hours after administration of about 29 mg/kg and high (given as DP) AVM0703 in naive Balb/c mice

Balb/c mice have MHC haplotype “d”: H-2K is d (H-2K _d ). H-2D is d (H-2D _d ). H2-L is d (H-2L _d ). Aαβ is d, d . Eαβ is d, d . Mls1 is b . Mls 2 is a . IA is d (IA _d ). IE is d (IE _d ). Qa-1 is b (Qa-1 _b ). Qa-2 is a (Qa-2 _a ).

C57Bl/6 mice have MHC haplotype "b": H-2K is b (H-2K _b ). H-2D is b (H-2D _b ). H2-L is null l . Aαβ is b and b . Eαβ is b and b . Mls1 is b . Mls 2 is b . IA is b (IA _b ). IE is null . Qa-1 is b (Qa-1 _b ). Qa-2 is a (Qa-2 _a ).

AVM NKT induced in naive Balb/c mice is CD3 MFI high, similar to peripheral blood AVM-NKT induced in naive C57Bl/6 mice, and AVM-NKT in naive Balb/c mice is TCRgamma/delta positive. NKp46 negative indicating that many cells were not activated. This example demonstrates that MHC expression can determine target organs.

MHC can control the trafficking of AVM_NKT cells: AVM_NKT cells are in the blood of naive AVM0703-treated male C57B16 mice. AVM_NKT cells are in the adipose and bone marrow of naive AVM0703 treated male Balb/c mice. AVM_NKT cells are present in the tumors of AVM0703-treated male tumor-bearing Balb/c mice. Novel NKTs in naive Balb/c mice are also tCRgd positive, B220-, NKp46+/-, Ly6G-, CD4-, CD8-, CD3high, MFI 10492, and CD49b+.

Example 6 Characterization of AVM-NKT

Early studies found that AVM-NKT cells appeared about 48 hours after treatment in the peripheral blood of animals treated with high doses of glucocorticoid receptor agonists (eg dexamethasone and betamethasone). A novel population of AVM-NKT cells induced by high doses of glucocorticoid receptor agonists when examined by flow cytometry was found to be CD49b+ and CD3 very bright (CD3highCD49b+). In contrast, previously described NKT cells express CD3 with one log lower MFI than AVM-NKT cells (CD3medCD49b+; FIG. 8 ).

In a subsequent experiment, C57Bl/6 animals were treated with high-dose dexamethasone (15 mg/kg HED dexamethasone base) and peripheral blood was examined by flow cytometry at predetermined time intervals to reveal different immune populations, and particularly new populations of AVM-NKT cells. characterized.

CD4

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD4 very bright (CD4high). CD4 median fluorescence intensity is higher than CD4 MFI for NKT events typical of other CD4+ T cells. CD4 MFI remains constant from 6 hours to 13 days after 15 mg/kg HED dexamethasone base ( FIG. 16 ).

CD8

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD8 dark. CD8+ MFI is not 1 log higher than typical NKT cells at the 6 h time point and drops linearly over the next 5 days. Less than 50% remain CD8+ at 72 hours and 5 days, but recover CD8+ on days 7-13 after 15 mg/kg HED dexamethasone base ( FIG. 17 ).

The majority of AVM-NKT cells are CD4 and CD8 double positive, unlike typical NKT, which are not double positive (Figs. 12, 13, 14). None of the AVM NKT cells are double negative for CD4 and CD8 ( FIG. 14 ). Known NKT cells (CD3med) are usually double negative or CD4+, some are CD8+ cells ( FIG. 14 ). For these known NKT cells, CD4 and CD8 expression patterns do not change over time after dexamethasone base.

AVM-NKT appears to be CD4+CD8+ at 48 h after 15 mg/kg HED dexamethasone base, loses CD8 positivity over time and then becomes CD4+CD8+ again later. They were evident 48 hours after AVM0703 and were detected on day 13.

CD3

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD3 very bright (CD3high), about 1 log higher than known NKT cells described in the literature and about 1 log higher than other NKT cells evident in C57Bl/6 male mice. CD3 is expressed with MFI ( FIG. 15 ).

Ly6G

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are Ly6G positive ( FIG. 18 ). Ly6G is a marker for fully mature and differentiated neutrophils or granulocytes and has also been implicated in antitumor responses. Ly6G is a marker for monocytes and neutrophils and granulocytes in general, which distinguishes AVM-NKT from known NKT cells, can directly kill cancer cells expressing CD1d, as well as activate other NK cells and B and T lymphocytes and It not only secretes cytokines, but also directly swallows cancer cells and pathogens.

TCR Gamma/Delta

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are TCR gamma delta positive.

Expression of Ly6G and TCR gamma delta suggests that AVM-NKT cells can directly engulf cancer cells or pathogens in addition to the known functions of NKT cells.

CD45

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD45 dark.

CD49b

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD49b positive. CD49b is a marker of natural killer (NK) cells; The cytotoxicity of NK cells expressing CD49b is much greater than that of NK cells not expressing CD49b.

CD62L

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD62L positive.

NK1.1

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists in C57Bl/6 mice are NK1.1 positive. NK1.1 is a marker of mature NK cells; Its activation induces NK cells to kill otherwise insensitive targets and can also induce proliferation of NK cells.

Sca1

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are Sca1 very bright. Sca1 (Ly6A), along with other markers, is a common biological marker used to identify hematopoietic stem cells (HSCs). Their bright expression on AVM-NKT cells may indicate that they are activated memory stem cells.

C-kit

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are C-kit negative. Therefore, they are not hematopoietic stem cells.

B220

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are B220 negative. B220 is a marker of B cells.

FoxP3

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are A FoxP3 negative. FoxP3 is a marker for regulatory cells - therefore, AVM-NKT should not attenuate the immune response of regulatory cells to cancer or pathogens.

TCR Alpha/Beta

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are TCR alpha/beta negative.

CD44+/-, CD69+/-, CD25+/-

AVM-NKT cells induced by high doses of glucocorticoid receptor agonists are CD44 +/-, CD69 +/-, and CD25 +/-.

CD44 expression is an indicator marker for effector-memory T-cells. CD69 and CD25 are markers of cell activation.

Based on the above characterization experiments, AVM-NKT cells induced by high-dose glucocorticoid receptor agonists engulf cancer cells and pathogens (they are Ly6G and TCR gamma delta positive), and cancer cells and other present in lipids through CD1d expression. It appears to be an activated effector memory stem cell type that can kill cells directly and have the ability to function as long-term T lymphocytes of both CD4 and CD8 strains. In addition, they can rapidly release cytokines in response to cancer cells or pathogens that can activate other cells important for the immune response.

The fact that AVM-NKT cells appear to add the potential to directly engulf cancer cells expands the potential of high-dose glucocorticoid receptor agonists as solid cancer therapeutics. As an off-the-shelf allogeneic therapeutic agent, alone or in combination with NKT activators or checkpoint inhibitors, or as AVM-NKT-CARs, AVM-NKT cells can be widely applied in the treatment of solid tumors. Furthermore, since AVM-NKT cells are not observed until after AVM0703 treatment, AVM-NKT levels may not limit treatment as low levels of NKT in the elderly and cancer patients limit the use of iNKT for self-treatment.

Example 7 - Acute High Dose Dexamethasone Has Tumor Killing Effect in T Cell and B Cell Lymphoma

High-dose dexamethasone was shown to significantly retard tumor growth in an A20 B-cell lymphoma tumor model ( FIG. 11 ). Subsequent dosing experiments were performed to investigate the optimal dosing schedule for AVM-NKT generation and tumor killing effects in the A20 B-cell lymphoma tumor model and the xenograft model of T-cell lymphoma (CCRF-CEM). The tested dosing schedules are summarized in Table 3 below.

Table 3: Exemplary dosing schedules

Example 8 - AVM-NKT cells are responsible for T and B leukocyte ablation in vivo.

Blood and spleen were collected from naive C57Bl6 mice. Mononuclear cells were isolated from blood and unicellular splenocytes were isolated from the spleen. Cells were incubated for 72 hours to a concentration of up to 500 micromolar dexamethasone phosphate, but in vitro apoptosis was not observed. Thus, complete in vivo T and B leukocyte ablation appears to be mediated by AVM-NKT and not by receptor-based mechanisms such as activation of the glucocorticoid receptor.

Example 9 - AVM-NKT cells are isolated and expanded and then used to precondition patients prior to cell therapy.

Autologous or allogeneic AVM-NKT cells are administered IV or IP to the patient 6 to 96 hours before cell therapy is administered. Cell therapy may be for regenerative purposes, to treat cancer, to treat an autoimmune disease or to treat an infection or other medical condition that requires cell therapy.

Example 10 - AVM-NKT induces oncolytic syndrome

AVM-NKT targets the tumor, forming a band of attack cells that invade the tumor like an army from all sides. Oncolytic syndrome develops and, in mice, is incurable and can lead to death. Clinical chemistry markers of oncolytic syndrome, such as uric acid, are elevated. A full examination of the tumor reveals a sludge-like oil encased in the tumor membrane.

Example 11 - AVM-NKT cells are used to prepare patients for cancer or other serious medical treatment

Autologous or allogeneic AVM-NKT cells are administered IV or IP to patients with performance conditions that do not receive medical treatment, such as chemotherapy, cell therapy, or organ or bone marrow transplantation. The patient's performance status is improved so that he can receive medical treatment.

Example 12 - AVM-NKT cells cause tumor pseudoprogression

Tumors treated with AVM-NKT cells appear to continue to grow, however, growth is inhibited due to tumor pseudoprogression due to AVM-NKT cells attracting the tumor through the release of cytokines and chemokines or direct binding of other immune cells. to be. Eventually, the tumor becomes completely acellular and is resorbed.

Example 13 - AVM-NKT cells are used to treat any type of cancer, graft versus host disease, autoimmune, or immune related adverse events of immunotherapy

AVM-NKT cells target both hematological and solid cancers, and fibroma tumors, benign tumors, and autoreactive T and B lymphocytes.

Example 14 - Acute High Dose Glucocorticoid Receptor Agonists Also Induce Unique T Cell Populations and Dendritic Cells

In addition to the novel populations of NKT cells (AVM-NKT) described in Examples 1-6, the authors found that high-dose dexamethasone was very high in CD3 T cells (AVM-T cells; FIG. 20), and CD11b very high dendritic cells (AVM). -dendritic cells; Figure 21) showed recruitment of a new population.

AVM-T cells induced by high doses of glucocorticoid receptor agonists are CD3 very bright (CD3high). Like novel AVM-NKT cells, novel AVM-T cells express 1-1.5 log higher CD3 than typical T or NKT cells ( FIG. 20 ). AVM-T cells induced by high doses of glucocorticoid receptor agonists are CD4 positive. AVM-T cells induced by high doses of glucocorticoid receptor agonists are CD45 dark. AVM-T cells induced by high doses of glucocorticoid receptor agonists are CD49b positive (human CD56 positive). AVM-T cells induced by high doses of glucocorticoid receptor agonists are CD8 positive.

AVM-dendritic cells induced by high doses of glucocorticoid receptor agonists are CD11b very bright (CD11b very high). CD11b very high AVM-dendritic cells express about 1 log higher CD11b than conventional CD11b+ dendritic cells. High-dose dexamethasone also increases the concentration of pre-existing CD11b+ dendritic cells in the blood ( FIG. 21 ).

High-dose glucocorticoids (dexamethasone) recruit novel CD3 very high natural killer T cells (AVM-NKT), novel CD3 very high T cells (AVM-T cells), and CD11b very high dendritic cells (AVM-dendritic cells) Therefore, it is believed that high-dose glucocorticoids such as dexamethasone and betamethasone will have clinical utility in the treatment of cancer, autoimmune diseases, and infectious diseases.

The data presented in Examples 14 and 15 demonstrate that the novel AVM-NKT significantly affects tumors for tumor killing and is effective in cancer models in which checkpoint inhibitors have been shown to be ineffective. Because AVM-NKT cells are recruited only after AVM0703 treatment (in contrast to other NK and NKTs that continuously circulate), AVM-NKT numbers may not be limited in patients.

iNKT cells have been shown to reduce influenza A-mediated inflammation and disease severity, and CD11b+ DCs are implicated in protection against respiratory syncytial virus and influenza A (H1N1). High-dose glucocorticoids, such as dexamethasone and betamethasone, are likely to be effective given that CD3 very high NKT and T cells are recruited, as cells with low cell CD3 and low CD11b expression levels are known to be effective against them. This is because it not only increases the number of dendritic cells, but also recruits CD11b dendritic cells with very high expression, which is not normally observed.

Example 15 - Acute High Dose Dexamethasone Reduces Tumor Volume and Improves Overall Survival in A20 Model of B Cell Lymphoma

The mouse A20 lymphoma model involves multiple direct (expression of the immunosuppressive molecules PD-L1, IDO, and IL-10, and lack of expression of the CD80 costimulatory molecule) and indirect (downregulation of APC function and induction of Treg cells) immune evasion. Because it uses a mechanism, it is a very aggressive tumor model. Also, in the study described below, AVM0703 was not administered until A20 tumors were very well established in ˜120 to 400 mm ³ volumes.

Male BALB/c mice were subcutaneously inoculated in the flank with A20 B lymphoma cells embedded in Matrigel. Tumor volume was monitored by caliper measurement, and when tumors were well established at about 150 mm ³ , or very well established at about 400 mm ³ for study "AVM_CANMOD_05", mice were treated at 7, 18, 22, or 25 mg Treated with AVM0703 at a HED dose of /kg. The endpoint is generally defined as a tumor volume of 1500 mm ³ .

Necrosis by HistoTox Labs (Boulder, CO) or tumor analysis that was scored blindly for viable tumors using MetaMorph analysis or microscopic brightfield evaluation showed that some AVM0703-treated mice were completely necrotic despite measurable tumor volume and even showed that they had fully resorbed tumors. Blindly scored necrosis in HistoTox Labs was significantly higher when 18, 22, and 25 mg/kg dose results were combined, and also significantly higher when 22 and 25 mg/kg AVM0703 treated mice were analyzed separately.

Some AVM0703-treated mice had no measurable tumors at study end, tumors were either completely necrotic or resorbed by MetaMorph or microscopic brightfield examination, or tumors had a maximal necrosis score of 5 in HistoTox Labs. These mice were pooled and contingency table analysis was performed using Fisher's exact test. Of the 52 mice treated with AVM0703 at 18-25 mg/kg, 10 had a complete response according to previous criteria; compared to 0 of 21 placebo-treated mice.

Taken together, these data indicate that AVM0703 has significant efficacy against aggressive lymphoma in HEDs greater than or equal to 18 mg/kg (Tables 4 & 5). AVM0703 treatment was also found to show significant inhibition of human T cell lymphoma CCRF-CEM growth in a pilot xenograft model (see Example 15).

Table 4: Summary of the A20 B-cell lymphoma model

Table 5: Individual study results for necrosis, percent dead area, live area, and microscopic necrosis/percent cell-free

AVM_CANMOD_01

In the first study ("AVM_CANMOD_01"), the effect of AVM0703 on the ability to reduce tumor volume and on overall survival was investigated in BALB/c mice (11 weeks of age) with well-established subcutaneous A20 tumors. Mice were randomized into two groups and administered orally with 18.06 mg/kg AVM0703 HED DP (n = 5) or placebo (n = 4) on days 7, 10, 18, 23, 24, 29, 36, and 43 post challenge. dealt with

Of the 5 AVM0703 treated mice, 4 mice each reached the endpoint after 7 doses and 1 mouse reached the endpoint after 8 doses. The study endpoint was defined as a tumor volume of 1500 mm ³ or a weight loss of at least 20%. With 8 doses of 18.06 mg/kg each, the total dose received by the latter mice was 145 mg/kg HED within 36 days. When the mice reach the endpoint, they are euthanized and the organs (colon, spleen, pancreas, and thymus) are examined and weighed during necropsy.

Tumor growth was delayed in AVM0703 treated mice ( FIG. 22 ). Mice treated with AVM0703 took approximately 2-fold longer to reach the tumor volume endpoint than mice treated with placebo. AVM0703-treated mice had a median time to termination of 41 days, whereas mice receiving only placebo had a median time from the first day of dosing to termination of 22 days ( FIG. 23 ).

Microscopic analysis showed significant differences in tumor structure in AVM0703 treated mice compared to placebo. The placebo tumor has an open structure and shows clear cellularity inside, suggesting that the tumor cells are concentrated in the center of the tumor. Tumors treated with AVM0703 had a more compact structure, showing extensive areas of necrosis. There were no cells in the center of the treated tumors ( FIG. 23 ).

One mouse in the AVM treatment group reached the 20% weight loss threshold and was euthanized after 46 days and 8 doses of AVM0703 ( FIG. 24 ). Group organ weight-to-body weight ratios were not significantly different at study endpoints for pancreas, thymus, and spleen; However, in the AVM0703 treatment group, the colon to body weight ratio increased slightly. Since AVM0703-treated mice reached the study endpoint between 14 and 40 days after placebo-treated mice reached the study endpoint, the increase in colonic weight may be due to the increasing age of the mice ( FIG. 25 ).

To determine whether changes in the spleen weight or thymus weight-to-body weight ratio in each mouse could indicate a sustained responsiveness to AVM0703 with repeated dosing, the data were based on the number of days post-AVM0703 administration and the total number of AVM0703 administrations received before the endpoint was reached. separated into

The results show that both the thymus and spleen remain responsive to repeated AVM0703 administrations for 7 doses, and lose responsiveness at 8 doses ( FIG. 26 ). On days 1 and 3 after the 7th dose of AVM0703, both spleen and thymus weights appear to have decreased and, as expected, recovered after AVM0703 administration.

AVM_CANMOD_03

In the second study ("AVM_CANMOD_03"), concentrations of AVM0703 HED DP (7, 18, and 26 mg/kg) in BALB/c mice (n = 5 per group) at 9, 16, 24, and 31 days after tumor inoculation were measured. administered in increasing doses. As in study AVM_CANMOD_01, treatment was started when the tumor was approximately 500 mm ³ in contrast to a well-established tumor of 150 mm ³ .

Similar efficacy was observed in the placebo and 7 mg/kg groups. The 18 and 26 mg/kg groups showed similar efficacy and some separation from the low-dose and placebo groups. No significant differences were found in spleen, thymus, colon, or pancreatic weights between groups of mice upon euthanasia. The endpoint curves showed no significant difference in median survival, although mice treated with 18 and 26 mg/kg had about 6 days more median survival than placebo-treated mice. There was no sudden death of the rat.

Based on evidence of pseudogrowth in tumors of AVM0703-treated mice in study AVM_CANMOD_01, tumors (n = 2 per group) were sent to HistoTox Labs for slicing and immunohistochemistry. Hematoxylin and eosin staining identified multiple areas of necrosis and slides were scored from 0 to 5 according to severity. Tumors from mice treated with AVM0703 had wider areas of necrosis, and mice administered 18 mg/kg DP had the greatest degree of necrosis as reflected in the scoring graph ( FIGS. 27C and 27E ).

CD3 expression was highest in placebo mice and visually decreased with increasing DP concentration ( FIG. 28 ). When staining the NK cell marker NKp46, there was a visual increase in cell staining with increasing DP concentration. In placebo-treated tumors, NK cells were concentrated around the blood vessels of the tumor ( FIG. 29A ). However, upon AVM0703 treatment, NK cells were localized at the edge between tumor growth and necrotic areas. From this, we concluded that NK cells contribute to necrotic expansion in tumors. Tumors stained for the NK cell marker CD49b had high background and staining of the epithelial tissue surrounding the tumor microvessels. As the dose of AVM0703 increased, the staining of the round cells indicated by the black arrows visually decreased ( FIG. 30 ). Finally, tumor slices were stained for caspase 3, an apoptotic cell marker. Apoptosis staining was increased at all doses of AVM0703 compared to placebo. This includes strong staining for apoptosis around areas of necrosis and isolated apoptosis in areas of neoplastic growth ( FIG. 31 ).

Taken together, treatment with AVM0703 increased NKp46-expressing cells, which were most likely to localize and contribute to intratumoral necrosis. Activated NKT cells are known to lose both CD3 and CD49b expression, and thus the elevated NK activity is most likely a combination of NK and NKT cell infiltration of the tumor. AVM0703 also induced increased apoptosis in tumors ( FIG. 31 ). This indicates that AVM0703 may induce more than one mechanism of tumor death.

AVM_CANMOD_04

In a third study ("AVM_CANMOD_04"), BALB/c mice with established A20 tumors were administered both AVM0703 and chemotherapy (cyclophosphamide/fludarabine [Cy/Flu]). Mice were randomized into the following groups (n = 3 to 5 per group): 1) placebo; 2) AVM0703 18 mg/kg HED DP Oral (PO) (Days 11 and 14); 3) Cy/Flu (days 11 and 14) [13.5 mg/kg/0.8 mg/kg, intraperitoneal (IP)/IP]; 4) AVM0703 18 mg/kg HED DP PO (day 11) followed by Cy/Flu [13.5 mg/kg/0.8 mg/kg, IP/IP] (day 14).

Tumor growth was reduced in mice administered twice with Cy/Flu and mice administered with Cy/Flu once combined with AVM0703 once. Combination-treated mice had a median time to endpoint of 73 days post tumor inoculation, whereas mice in the Cy/Flu group did not reach endpoint at study end point (95 days post tumor inoculation).

The tumors in this study were paraffin-embedded and excised. Two section images of each tumor were transferred to the AVM. Images of tumor sections were subsequently uploaded to MetaMorph image analysis software. The percentage of dead tumors was determined using Image Thresholding software. The viable tumor area was subsequently calculated by subtracting the critical area from the total tumor area. All tasks were performed blinded to the group to which the image belonged.

For the Cy/Flu groups administered on days 11 and 14, 2 of 3 mice had no tumors at the endpoint; However, the third mouse clearly relapsed and had a very large tumor with a viable tumor area of 169,362 random units (AU). One mouse in the AVM0703 (Day 11) and Cy/Flu (Day 14) combination group had tumors, which were completely resorbed ( FIG. 32 ) but with an area of tumors of 182,279 AU, an example of pseudo-progression.

Two of the other mice in the combination group had tumors that were 90% necrotic and had only viable tumor areas between 10,000 and 25,000 AU. The mean viable tumor area for 5 mice in the combination group was only 16,490 AU, compared to the mean viable tumor area of 104,318 AU for the placebo group, or 182,279 AU for the viable tumor area for relapsed Cy/Flu mice. In this small study, the 18 mg/kg AVM0703 group had a smaller viable tumor volume (94,305 AU), but this was not significantly different from placebo mice.

When compared to published results with CHOP (cyclophosphamide, hydroxydaunorubicin oncobin, prednisone) chemotherapy in the A20 model, AVM0703 in combination with Cy/Flu resulted in a longer remission than 1 cycle of CHOP chemotherapy. induced, maintained remission longer than 2 cycles of CHOP chemotherapy, and tumor prolapse was observed at approximately day 42.

AVM_CANMOD_05

A fourth study ("AVM_CANMOD_05") was conducted to investigate how high doses (18, 22, and 25 mg/kg HED DP) of AVM0703 affect antitumor capacity in the A20 B cell lymphoma mouse model.

The study AVM_CANMOD_05 was divided into two subsets: lymph depletion and endpoint analysis. A previous in-house lymphatic depletion study was performed in naive C57BL/6 mice, demonstrating the lymphatic depleting effect of AVM0703 in healthy mice. In order to better understand the previous data suggesting the anti-tumor effect of AVM0703 and to better understand the mechanism of action of AVM0703 in tumor models, it was necessary to elucidate the profile of lymphatic depletion in in vivo tumor models.

T lymph-depleted subset mice were euthanized 48 hours after administration. The second subset, endpoint analysis, focused on examining the effect of repeated high doses of AVM0703 on time to endpoint in study mice. Importantly, mice in this study were dosed when the A20 tumor was very large, about 390 mm ³ .

Checkpoint inhibitors such as anti-PD-1 (eg KEYTRUDA) are approved for clinical use in B cell lymphoma. In this highly aggressive A20 B-cell lymphoma model, anti-PD 1 is not effective if treatment is started after tumors have reached ≥100 mm ³ . Anti-PD 1 is effective in this model only if treatment is started within 3 days of A20 inoculation, before tumors are palpable.

HistoTox Labs scored the lymphatic depletion subset. In the lymph-depleted subset, 2 out of 9 mice scored 5 for necrosis (range 0 to 5). One in 23 mice in the endpoint analysis subset showed complete tumor death and resorption, resulting in an overall complete response rate of 9%—better than a 0% complete response rate to anti-PD-1 for established A20 tumors.

Tumor analysis using MetaMorph image analysis software demonstrated that 18 and 25 mg/kg AVM0703 treated mice had greater tumor resorption than placebo treated mice ( FIG. 33 ). However, in this study, tumors were prolapsed and growing except for one mouse treated with 18 mg/kg where the tumors were 99.5% resorbed. A published study using the A20 model in an AVM-like aggressive manner showed that even 2 cycles of CHOP, which directly killed 18% of mice, showed 100% relapse within 20 days of complete remission.

The HistoTox Labs score of the T lymphodepletion subset showed increased tumor necrosis (hematoxylin and eosin), decreased CD3 & CD49b labeling, which may represent activated immune cells, and increased Ly6G expression (of AVM-NKT cells) in tumors of AVM0703-treated mice. marker) was demonstrated. For the endpoint analysis subset, tumors of AVM0703-treated mice reduced CD3 and CD49b labeling, increased tissue of NKp46 cells (NK and NKT cells), and decreased Ly6G, Sca1, and collagen labeling. In the lymph-depleted subset, ALC was inversely proportional to AVM0703 dose. At the 22 mg/kg and 25 mg/kg HED DP doses, the unresected lymphocytes were predominantly NK and NKT cells, and B cells. The 18 mg/kg HED DP dose almost completely eliminated B cell lymphocytes but not NK and NKT cells. The different lympholysis profiles may be due to mouse strain differences in sensitivity to AVM0703 or may be due to differences between naive and tumor models.

Importantly, in the tumor model, glucose levels were not elevated in contrast to observations made in naive C57BL/6 mice. At 18 mg/kg HED, glucose levels did not reach hypoglycemic levels but decreased significantly.

Example 16—Acute High Dose Dexamethasone Has Inhibitory Effect in CCRF-CEM Human T Cell Lymphoma Xenograft Model

A pilot study (“AVM_CANMOD_06”) was conducted to investigate the anti-tumor efficacy of AVM0703 in a human T cell lymphoma model, CCRF-CEM. Female NCr nude mice were inoculated with CCRF CEM human T-lymphoblasts and treated weekly with oral placebo (n = 2) or oral 18 mg/kg AVM0703 (n = 3) after a 7-day transplant period.

Tumor volume was assessed 3 times per week and endpoint was defined as tumor volume greater than 1500 mm ³ or greater than 20% loss at initial body weight measurement. Mice inoculated with CCRF-CEM cells exhibited a time delay to endpoint when treated with AVM0703 compared to placebo ( FIGS. 36 and 37 ). Overall, there is a tendency for tumor growth to be delayed in CCRF CEM tumor bearing mice treated with AVM0703 compared to placebo.

One mouse in the AVM0703 treatment group was found dead 89 days after tumor inoculation—the tumor was removed and photographed ( FIG. 35 ). Significant oncolysis is evident and is most responsible for the death of these mice. AVM0703-treated mice 3R were rechallenged (3L) with human T-ALL (CCRF-CEM cell line) on day 118 and there was no tumor growth until day 164 ( FIG. 38 ). Placebo mice reach a tumor volume endpoint of 1500 mm ³ on days 50-55. AVM0703 treated mice did not reach tumor volume endpoints.

Example 17—Identification of AVM-NKT Cells in Human Subjects Treated with Acute High Dose of Dexamethasone

Following identification of AVM-NKT cells in mice, data from files from human subjects treated with high doses of dexamethasone were reanalyzed.

In osteoarthritis patients, 3-6 mg/kg generic dexamethasone was administered to 4 patients according to the guidelines of "Physician Practice of Medicine" (Dr. Loniewski, Advanced Orthopedic Surgeon, Brighton, MI).

A review of flow cytometry data after 48 hours was performed from 4 patients treated with a dexamethasone dose 6-fold lower than the dose used to maximally induce AVM_NKT in mice. A CD45/CD56 scatterplot from one of four patients showed that a new cell population corresponding to the AVM-NKT cells identified in mice appeared ˜48 hours after treatment ( FIG. 39 ).

As shown in Figure 40, a novel CD56 very bright cell population was observed in prostate cancer patients 1 hour after their 4th AVM0703 treatment was injected at 6 mg/kg. The prostate cancer patient was an out-of-choice patient after many years of cancer treatment and received a total of 4 AVM0703 injections at least 28 days apart.

Compared with healthy donors, prostate cancer patients had evidence of a novel CD3 dark population that was no longer evident 1 hour after AVM0703, whereas a new CD56 very bright cell population was evident in the blood that was no longer observed 3 hours after injection. showed up

Compared with healthy donors, prostate cancer patients had CD3 dark and NKp46dim cell populations before injection, and 1 hour after injection of 6 mg/kg AVM0703, patients had a new CD56 very bright CD3dim population with CD45 dark/negative and CD4/CD8 double negative. have

Example 18 - Production and recruitment of human AVM-NKT cells in humanized mice

BRGSF humanized mice of Genoway's Balb/c background generated by transplantation of human cord blood CD34+ stem cells into irradiated mice lacking mouse B and T lymphocytes and NK cells but with a functional mouse complement system were HED 18-45 mg/kg. Administer orally with DSP. After 24-48 hours human CD3high, and/or human CD45dim, and/or human CD56+ cells can be observed to be about 0.2-3% of total splenocytes by flow cytometry. Human CD3high, human CD45dim, and human CD56+ cells can be observed in the blood from about 36 hours to 13 days later.

HuCD34-NCG mouse model

Charles River's HuCD34-NCG mouse is a research mouse model with a human-like immune system generated by adoptive transfer of CD34+ stem cells. The HuCD34-NCG mouse is an ideal in vivo platform for evaluating the effects of compounds that modulate the human immune system. Since graft-versus-host disease (GvHD) is absent or late onset in humanized mice, it is ideal for long-term studies.

NCG mice are humanized by adoptive transfer using human cord blood-derived CD34+ stem cells after bone marrow resection treatment. NCG from 4 donors (n=2 per donor) is administered orally at HED 18-45 mg/kg DSP. After 24-48 hours human CD3high, and/or human CD45dim, and/or human CD56+ cells can be observed to be about 0.2-3% of total splenocytes by flow cytometry. Human CD3high, human CD45dim, and human CD56+ cells can be observed in the blood from about 36 hours to 13 days later.

huNOG-EXL mouse model

Taconic's huNOG EXL has an average of 54% of CD45 cells positive for human CD45. Six huNOG EXL humanized immune system mice (n=2 per donor) from 3 donors are orally administered with HED 18-45 mg/kg DSP. After 24-48 hours human CD3high, and/or human CD45dim, and/or human CD56+ cells can be observed to be about 0.2-3% of total splenocytes by flow cytometry. Human CD3high, human CD45dim, and human CD56+ cells can be observed in the blood from about 36 hours to 13 days later.

Example 19 - Venetoclax pretreatment dose-dependently reduces the number of AVM-NKT cells recruited into blood after administration of high-dose dexamethasone

10-week-old female NOD mice were treated with venetoclax at 12.5 mg/kg up to 50 mg/kg 6-18 hours prior to oral gavage at a dose of 30 mg/kg DSP HED. Venetoclax pretreatment dose-dependently reduced the number of CD3high, CD45dim, and CD49b+ cells recruited into the blood for 48 hours after administration of 30 mg/kg DSP. CD3high, CD45dim, and CD49b+ cells increased from ~70 cells/microliter with DSP alone to ~40 cells/microliter with 12.5 mg/kg Venetoclax pretreatment and ~20 cells/microliter with 25 mg/kg Venetoclax pretreatment. , reduced to ~15 cells/microliter upon 50 mg/kg venetoclax pretreatment. Venetoclax is a Bcl-2 inhibitor.

Example 20 - Acute High Dose Dexamethasone Prevents or Delays Hyperglycemia in Female Spontaneous Diabetic NOD Mice

Female NOD mice were ordered at 9 weeks of age. At 10 weeks of age, with complete infiltration of insulitis in the pancreas, mice were dosed with placebo as appropriate for each treatment, or cyclosporine twice weekly at 5 mg/kg for 7 weeks, followed by cyclosporine twice weekly at 10 mg/kg for the remainder of the 5-month study; or single acute oral single dose dexamethasone (AVM0703) at 18 mg/kg or 30 mg/kg HED, or venetoclax at 25 mg/kg, or venetoclax at 25 mg/kg followed by 18-24 hours HED 30 mg/kg dexamethasone was administered.

Body weight and blood sugar levels were monitored weekly. Physical condition was monitored 3 times a week. Oral glucose tolerance was determined in all surviving mice reaching 30 weeks of age. The remaining mice were necropsied and autoreactive lymphocytes from the pancreas and adjacent lymph nodes were determined by flow cytometry. Pancreatitis was determined by H&E staining (8 out of 15 per group). Pancreatic beta cell surface area was measured by insulin staining. Insulin secreting islets were scored as follows: 1, no insulitis (no infiltration); 2, peripancreatitis (inflammatory cells outside or in the immediate vicinity of the islets); 3, Insulitis (clear and extensive islet infiltration showing direct contact with lymphocyte-beta cells). Autoreactive insulin-specific CD4+ T cells were tested in the pancreas and pancreatic lymph nodes using a magnetic enrichment method with tetrameric reagents.

AVM0703-treated mice performed significantly better than mice in all other groups throughout the 5-month study. Venetoclax alone accelerated the onset of diabetes, which was delayed when Venetoclax was administered followed by AVM0703. Alone, AVM0703 prevented diabetes in 40% of mice and significantly delayed the onset in the remaining 60% of mice. At the end of the 5-month study, mice treated with AVM0703 without hyperglycemia showed a normal oral glucose tolerance test (OGTT), whereas mice in all other groups had elevated glucose levels in response to fasting OGTT.

Example 21 - Acute High-Dose Dexamethasone Reverses Diabetes in Female NOD Mice with Established Diabetic Early onset

Female NOD mice are ordered at 9 weeks of age. Blood sugar levels are measured weekly starting at 10 weeks of age. If the mouse's non-fasting blood glucose is greater than 250 mg/dl, another measurement is performed the next day.

For reversal of newly onset diabetes, AVM0703 administration was started one day after the mice had elevated non-fasting blood glucose levels for two consecutive days. Insulin pellets are implanted subcutaneously on the second day when blood sugar levels rise. For reversal of established diabetes, elevated blood glucose was measured for two consecutive weeks, insulin pellets implanted on day 8 from the first day of measurement of elevated blood glucose level, and AVM0703 on day 14 from the first day of measured elevated blood glucose level dosing

Compared to anti-CD3 or ATG treated mice, AVM0703 can equally reverse early onset and established diabetes without the weight loss or poor physical condition observed in anti-CD3 or ATG treated mice.

references

Numerous publications have been cited above in order to more fully describe and disclose the present invention and the state of the art to which the present invention pertains. Each reference is incorporated herein by reference in its entirety. Full citations to these references are as follows:

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STATEMENT OF DISCLOSURE

The following numbered statements that summarize aspects of the present disclosure are part of the description.

AVM-NKT cells

101. A method of producing and/or recruiting a population of natural killer T cells (NKT cells), said method comprising administering to a subject a glucocorticoid receptor ( GR) administering a modulator or an ICAM3 modulator,

wherein the glucocorticoid receptor (GR) modulator or ICAM3 modulator induces and/or recruits a population of NKT cells in the subject.

NKT cell marker expression

102. The method of statement 101, wherein the population of NKT cells contains at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells:

i) express CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;

ii) does not express C-kit, B220, FoxP3, and/or TCR alpha/beta.

103. The method of statement 102, wherein said NKT cell expresses:

(i) CD3, CD4, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, and Sca1; or

(ii) CD3, CD45, and CD56.

104. The method of any of statements 102-103, wherein said NKT cells do not express C-kit, B220, FoxP3, or TCR alpha/beta.

105. The method according to any one of statements 102 to 104, wherein said NKT cells do not express CD8.

106. The NKT cell according to any one of statements 102 to 104, wherein:

i) express CD4 and CD8;

ii) a method of expressing Ly6G.

107. The NKT cell according to any one of statements 102 to 106, wherein:

i) CD4+/very bright;

ii) CD8+/dark;

iii) CD3+/very bright;

iv) CD45+/dark;

v) Sca1+/very bright;

vi) CD44+/-;

vii) CD69+/-;

viii) CD25+/-; and/or

ix) CD3+/very bright and CD45+dark and CD56+;

Optionally, wherein the expression level is determined relative to the mean expression level of a reference NKT cell population derived from a common source and not contacted with a glucocorticoid receptor (GR) modulator.

108. The method of any one of statements 102-107, wherein said expression is measured by flow cytometry, optionally wherein said flow cytometry is performed using the equipment, reagents and/or conditions described herein (taken alone or in combination). how it is.

glucocorticoids

109. The method of any one of statements 101 to 108, wherein the glucocorticoid receptor (GR) modulator is a glucocorticoid, optionally wherein the glucocorticoid is selected from the group consisting of: dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide, betamethasone, flumethasone and beclomethasone.

110. The method according to statement 109, wherein said glucocorticoid is selected from the group consisting of dexamethasone, betamethasone, and methylprednisone, preferably said glucocorticoid is dexamethasone or betamethasone.

111. The method of any one of statements 108 to 110, wherein the glucocorticoid is dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicotinate, dexamethasone-21-acetate, Dexamethasone phosphate, dexamethasone-21-phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate, dexamethasone palmitate, dexamethasone-21-palmitate, dexamethasone dipropionate, dexamethasone dipropionate , dexamethasone acetate anhydrous, dexamethasone-21-phenylpropionate, dexamethasone-21-sulfobenzoate, dexamethasone hemo-sulfate, dexamethasone sulfate, dexamethasone beloxyl, dexamethasonic acid, dexamethasone acefurate, dexamethasone carboxylate, dexamethasone carboxylate , dexamethasone 21-phosphate disodium salt, dexamethasone mesylate, dexamethasone linoleate, dexamethasone glucoside, dexamethasone glucuronide, dexamethasone iodine acetate, dexamethasone oxetanone, carboxymethylthio-dexamethasone, dexamethasone epoxetoxime, dexamethasone epoxide , dexamethasone linoleidate, dexamethasone methylorthovalerate, dexamethasone spermine, 6-hydroxy dexamethasone, dexamethasone tributyl acetate, dexamethasone aspartic acid, dexamethasone galactopyranose, dexamethasone hydrochloride, hydroxydexamethasone, carboxydexamethasone Desoxydexamethasone, dexamethasone butazone, dexamethasone cyclodextrin, dihydrodexamethasone, oxo-dexamethasone, propionyloxy dexamethasone, dexamethasone galactodi, dexamethasone isonicotinate, dexamethasone sodium hydrogen phosphate tridecyldecyldecyldecyldecide, dexamethasone Late, dexamethasone crotonate, dexamethasone methanesulfonate, dexamethasone butyl acetate, dehydro dexamethasone, dexamethasone isothiocyanatoethyl) thioether, dexamethasone bromoacetate, dexamethasone hemiglutarate, deoxy A method selected from the group consisting of dexamethasone, dexamethasone chlorambusylate, dexamethasone melpalanate, formyloxy dexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and any combination treatment containing the form of dexamethasone.

112. The method of statement 111, wherein said dexamethasone is dexamethasone sodium phosphate.

Glucocorticoid Dosage

113. The glucocorticoid of any one of statements 101 to 112, wherein the glucocorticoid is administered at a dose approximately equal to:

i) at least 6-12 mg/kg human equivalent dose (HED) of dexamethasone base;

ii) at least 6 mg/kg human equivalent dose (HED) of dexamethasone base;

iii) at least 12 mg/kg human equivalent dose (HED) of dexamethasone base;

iv) at least 15 mg/kg human equivalent dose (HED) of dexamethasone base;

v) at least 18 mg/kg human equivalent dose (HED) of dexamethasone base;

vi) at least 24 mg/kg human equivalent dose (HED) of dexamethasone base;

vii) 15 mg/kg human equivalent dose (HED) of dexamethasone base;

viii) 24 mg/kg human equivalent dose (HED) of dexamethasone base;

ix) 30 mg/kg human equivalent dose (HED) of dexamethasone base;

x) 45 mg/kg human equivalent dose (HED) of dexamethasone base; or

xi) The human equivalent dose (HED) of dexamethasone base takes a mg/kg value in a range of mg/kg values, wherein the range is limited by two of the mg/kg values given in parts i) to x) above.

114. The method of any one of statements 101 to 113, wherein the glucocorticoid is administered in a single acute dose, or in a total dose given over about 72 hours.

115. The method of any of statements 101-114, wherein the method comprises administering one or more additional doses of the glucocorticoid.

116. The method of statement 115, wherein the one or more additional doses are administered as follows:

i) 24 to 120 hours after prior glucocorticoid administration;

ii) 24 to 48 hours after prior glucocorticoid administration;

iii) 72 to 120 hours after prior glucocorticoid administration;

iv) every 24, 48, 72, 96, 120, 144, or 168 hours after the first glucocorticoid administration;

v) once every 2 weeks after the first glucocorticoid administration;

vi) once a month after the first glucocorticoid administration; or

vii) Twice weekly after the first glucocorticoid administration.

NKT cell activation

117. The method of any of statements 101-116, further comprising administering to the subject a NKT cell activator.

118. The method of statement 117, wherein said NKT cell activator is selected from the group consisting of alpha GalCer, sulfide, or NKT-activating antibody.

119. The method of statement 118, wherein the NKT cell activator is an alpha GalCer loaded dendritic cell or a monocyte.

120. The method of any one of statements 117 to 119, wherein the NKT cell activator is administered within 48 hours or before or after 48 hours after administration of the glucocorticoid.

object

121. The method according to any one of statements 101 to 120, wherein the subject is a mammal, preferably the subject is a human.

122. The method of any of statements 101-121, wherein the subject has, is suspected of having, or has been diagnosed with a disease selected from the group consisting of cancer, an autoimmune disease, or an infectious disease.

123. The method of statement 122, wherein said cancer is a solid tumor cancer.

124. The method of statement 122, wherein said cancer is selected from the group consisting of: squamous cell carcinoma (eg epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung; peritoneal cancer; hepatocellular carcinoma; stomach or abdominal cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver tumor; breast cancer; colorectal cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary adenocarcinoma; kidney or kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocarcinoma; anal carcinoma; penile carcinoma; and head and neck cancer.

125. The method according to statement 122, wherein the cancer is a lymphoma, preferably a B cell lymphoma, a T cell lymphoma, or a non-Hodgkin's lymphoma.

126. The method of any one of statements 122-125, wherein said NKT cells treat cancer through tumor infiltration.

127. The method of statement 126, wherein said NKT cells treat cancer through the release of immune activating cytokines.

128. The method of statements 126 or 127, wherein said NKT cells engulf and kill cancer cells.

129. The method according to any one of statements 126-128, wherein said NKT cells promote infiltration of other immune cells into the tumor.

130. The method according to any one of statements 126-129, wherein said NKT cells directly kill cancer cells via CD1d-induced apoptosis.

131. The method according to any one of statements 126-130, wherein said NKT cells cause tumor necrosis.

132. The method of statement 122, wherein the autoimmune disease is selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes mellitus (T1D), scleroderma, pemphigus, and lupus.

133. The method of statement 122, wherein the autoimmune disease is type 1 diabetes mellitus (T1D).

134. The method of statement 122, wherein the infectious disease is selected from the group consisting of HIV and a disease caused by a coronavirus infection such as herpes, hepatitis, human papillomavirus, or COVID-19.

135. The method of statement 122, wherein the infectious disease is:

i) HIV; or

ii) How it is COVID-19.

Isolation/Expansion Step

136. The method of any one of statements 101 to 135, further comprising isolating the population of NKT cells from the subject or from a sample derived from the subject,

Optionally, the isolating step is performed as follows:

i) at least 48 hours after administration of the glucocorticoid;

ii) 48 hours to 13 days after administration of glucocorticoids; or

iii) 6 to 48 hours after administration of the glucocorticoid.

137. The method of statement 136, wherein said sample is selected from the group consisting of blood, plasma, tumor biopsy or surgically removed tumor, bone marrow, liver, and adipose or adipose tissue.

138. The method of statements 136 or 137, further comprising expanding the isolated NKT cells.

139. The method according to any one of statements 136 to 138, further comprising activating the isolated NKT cells with a NKT cell activator.

Optionally, the NKT cell activator is selected from:

i) cytokines, chemokines, growth factors, and/or NKT modulators;

ii) alpha GalCer (alpha-galactosylceramide; α-GalCer) sulfatide (3-O-sulfogalactosylceramide; SM4; sulfated galactocerebroside).

Transfection of isolated NKT cells

140. The method of any one of statements 136 to 139, further comprising introducing a nucleic acid encoding the protein into the isolated NKT cell, and culturing the cell under conditions that promote expression of the protein.

141. The method of statement 140, wherein said protein is selected from the group consisting of one or more of T-cell receptor (TCR), chimeric antibody receptor (CAR), split, universal and programmable CAR (SUPRA-CAR).

142. The method of statement 141, wherein said CAR and/or TCR comprises an antigen binding domain that binds to an antigen selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1 , mesothelin, IL-13R, PSMA, ROR1, CAIX, Her2.

143. The method of any one of statements 140-142, further comprising expanding the NKT cells.

144. The method of any of statements 140-143, further comprising activating the NKT cells with an NKT cell activator.

Administration of isolated NKT cells

145. A method of treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising NKT cells isolated according to any one of statements 136 to 144 of a therapeutically effective dose, according to any one of statements 401 to 406 A method comprising administering to the subject the isolated NKT cells, or the cell population of statement 407.

146. The method of statement 145, wherein the subject to which the isolated NKT cells are administered is the same subject from which the NKT cells were isolated.

147. The method of statement 145, wherein the subject to which the isolated NKT cells are administered is different from the subject from which the NKT cells were isolated.

148. The method according to any one of statements 145 to 147, wherein said NKT cells are administered to the subject by a method selected from the group consisting of: intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, Injections in cerebrospinal fluid (CSF), direct injections into tumors, gels on or near solid tumors.

medical use

149. A glucocorticoid for use in a method according to any one of statements 101 to 148.

150. Use of a glucocorticoid for the manufacture of a medicament for use in a method according to any one of statements 101 to 148.

151. Use of dexamethasone for inducing and/or recruiting a population of NKT cells, wherein the population of NKT cells is derived and/or recruited by a method according to any one of statements 101 to 148.

AVM-NKT-derived iPSCs

152. A method of producing induced pluripotent stem cells (iPSCs), the method comprising reprogramming the NKT cells isolated by the method according to any one of statements 136 to 138 to produce iPSCs.

153. The method of statement 152, wherein said reprogramming comprises introducing into the NKT cell one or more expression cassettes encoding Oct3/4, Klf4, Sox2, and C-myc.

154. The method of statement 152, wherein said reprogramming comprises introducing Oct3/4, KLF4, Sox2, and c-myc encoding mRNA into the NKT cell.

155. The method of statements 153 or 154, wherein said reprogramming comprises generating one or more expression cassettes encoding one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28. A method further comprising introducing into NKT cells.

156. The NKT cell of statements 153 or 154, wherein said reprogramming introduces one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28 encoding mRNA into the NKT cell. A method comprising additional steps.

157. The method of any one of statements 152-156, further comprising inducing differentiation of iPSCs.

158. The method of statement 157, wherein said iPSCs differentiate into NKT cells.

159. A method of producing a population of NKT cells, said method comprising differentiating iPSCs produced by the method according to any one of statements 152-156 into a NKT cell lineage.

AVM-T cells and AVM-dendritic cells

160. The method of any one of statements 101 to 135, wherein the glucocorticoid receptor (GR) modulator also induces a population of T cells in the subject,

Optionally, said T cell is as defined by any one of statements 202-205.

161. The method of any one of statements 101 to 135 or 160, wherein the glucocorticoid receptor (GR) modulator also activates a population of dendritic cells in the subject,

Optionally, the dendritic cell is as defined by any one of statements 302-304.

- - -

AVM-T cells

201. A method of producing a population of T cells, the method comprising administering to a subject a glucocorticoid receptor (GR) modulator or an ICAM3 modulator at a dose equivalent to approximately at least 6 mg/kg human equivalent dose (HED) of dexamethasone base. including,

wherein the glucocorticoid receptor (GR) modulator or ICAM3 modulator induces a population of T cells in a subject.

T cell marker expression

202. The method of statement 201, wherein said population of T cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells:

i) express CD3, CD4, CD45, and/or CD49b;

ii) does not express CDCD49b.

203. The method of statement 202, wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the population of T cells express TCR gamma/delta.

204. The method of any one of statements 202-203, wherein the T cell is CD3+/very bright,

Optionally, wherein the expression level is determined relative to the average expression level in a reference T cell population derived from a common source, not contacted with a glucocorticoid receptor (GR) modulator.

205. The method of any one of statements 202-204, wherein said expression is measured by flow cytometry, optionally wherein said flow cytometry is performed using the equipment, reagents and/or conditions described herein (taken alone or in combination). How to be.

glucocorticoids

206. The method of any one of statements 201 to 205, wherein the glucocorticoid receptor (GR) modulator is a glucocorticoid, optionally wherein the glucocorticoid is dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide , betamethasone, flumethasone and beclomethasone.

207. The method of statement 206, wherein the glucocorticoid is selected from the group consisting of dexamethasone, betamethasone, and methylprednisone, preferably the glucocorticoid is dexamethasone or betamethasone.

208. The method of any one of statements 206 to 207, wherein the glucocorticoid is dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicotinate, dexamethasone-21-acetate, Dexamethasone phosphate, dexamethasone-21-phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate, dexamethasone palmitate, dexamethasone-21-palmitate, dexamethasone dipropionate, dexamethasone dipropionate , dexamethasone acetate anhydrous, dexamethasone-21-phenylpropionate, dexamethasone-21-sulfobenzoate, dexamethasone hemo-sulfate, dexamethasone sulfate, dexamethasone beloxyl, dexamethasonic acid, dexamethasone acefurate, dexamethasone carboxylate, dexamethasone carboxylate , dexamethasone 21-phosphate disodium salt, dexamethasone mesylate, dexamethasone linoleate, dexamethasone glucoside, dexamethasone glucuronide, dexamethasone iodine acetate, dexamethasone oxetanone, carboxymethylthio-dexamethasone, dexamethasone epoxetoxime, dexamethasone epoxide , dexamethasone linoleidate, dexamethasone methylorthovalerate, dexamethasone spermine, 6-hydroxy dexamethasone, dexamethasone tributyl acetate, dexamethasone aspartic acid, dexamethasone galactopyranose, dexamethasone hydrochloride, hydroxydexamethasone, carboxydexamethasone Desoxydexamethasone, dexamethasone butazone, dexamethasone cyclodextrin, dihydrodexamethasone, oxo-dexamethasone, propionyloxy dexamethasone, dexamethasone galactodi, dexamethasone isonicotinate, dexamethasone sodium hydrogen phosphate tridecyldecyldecyldecyldecide, dexamethasone Late, dexamethasone crotonate, dexamethasone methanesulfonate, dexamethasone butyl acetate, dehydro dexamethasone, dexamethasone isothiocyanatoethyl) thioether, dexamethasone bromoacetate, dexamethasone hemiglutarate, deoxy A method selected from the group consisting of dexamethasone, dexamethasone chlorambusylate, dexamethasone melpalanate, formyloxy dexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and any combination treatment containing a form of dexamethasone.

209. The method of statement 208, wherein said dexamethasone is dexamethasone sodium phosphate.

Glucocorticoid Dosage

210. The glucocorticoid of any one of statements 201 to 209, wherein the glucocorticoid is administered at a dose equivalent to:

i) at least 6-12 mg/kg human equivalent dose (HED) of dexamethasone base;

ii) at least 6 mg/kg human equivalent dose (HED) of dexamethasone base;

iii) at least 12 mg/kg human equivalent dose (HED) of dexamethasone base;

iv) at least 15 mg/kg human equivalent dose (HED) of dexamethasone base;

v) at least 18 mg/kg human equivalent dose (HED) of dexamethasone base;

vi) at least 24 mg/kg human equivalent dose (HED) of dexamethasone base;

vii) 15 mg/kg human equivalent dose (HED) of dexamethasone base;

viii) 24 mg/kg human equivalent dose (HED) of dexamethasone base;

ix) 30 mg/kg human equivalent dose (HED) of dexamethasone base;

x) 45 mg/kg human equivalent dose (HED) of dexamethasone base; or

xi) The human equivalent dose (HED) of dexamethasone base takes a mg/kg value in a range of mg/kg values, wherein the range is limited by two of the mg/kg values given in parts i) to x) above.

211. The method of any of statements 201 to 210, wherein the glucocorticoid is administered in a single acute dose, or in a total dose given over about 72 hours.

212. The method of any of statements 201 to 211, wherein the method comprises administering one or more additional doses of the glucocorticoid.

213. The method of statement 212, wherein the one or more additional doses are administered as follows:

viii) 24 to 120 hours after prior glucocorticoid administration;

ix) 24 to 48 hours after prior glucocorticoid administration;

x) 72 hours to 120 hours after prior glucocorticoid administration;

xi) every 24, 48, 72, 96, 120, 144, or 168 hours after the first glucocorticoid administration;

xii) once every 2 weeks after the first glucocorticoid administration;

xiii) once monthly after the first glucocorticoid administration; or

xiv) Twice weekly after the first glucocorticoid administration.

T cell activation

214. The method of any one of statements 201 to 213, further comprising administering to the subject a T cell activator.

215. The method of statement 214, wherein the T cell activating agent is a T cell activating antibody.

216. The method of any one of statements 214 to 215, wherein the T cell activator is administered within 48 hours or within about 48 hours after administration of the glucocorticoid.

object

217. The method according to any one of statements 201 to 216, wherein the subject is a mammal, preferably the subject is a human.

218. The method of any of statements 201 to 217, wherein the subject has, is suspected of having, or has been diagnosed with a disease selected from the group consisting of cancer, an autoimmune disease, or an infectious disease.

219. The method of statement 218, wherein said cancer is a solid tumor cancer.

220. The method of statement 218, wherein said cancer is selected from the group consisting of: squamous cell carcinoma (eg epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung; peritoneal cancer; hepatocellular carcinoma; stomach or abdominal cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver tumor; breast cancer; colorectal cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary adenocarcinoma; kidney or kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocarcinoma; anal carcinoma; penile carcinoma; and head and neck cancer.

221. The method according to statement 218, wherein said cancer is a lymphoma, preferably a B cell lymphoma, a T cell lymphoma, or a non-Hodgkin's lymphoma.

222. The method according to any one of statements 218 to 221, wherein said T cells are through tumor infiltration. How to treat cancer.

223. The method of statement 222, wherein the T cell is activated through the release of an immune activating cytokine. How to treat cancer.

224. The method according to any one of statements 222 to 223, wherein said T cells promote infiltration of other immune cells into the tumor.

225. The method according to any one of statements 222 to 224, wherein said T cells directly kill cancer cells by inducing apoptosis.

226. The method according to any one of statements 222 to 225, wherein said T cells cause tumor necrosis.

227. The method of statement 218, wherein said autoimmune disease is selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes mellitus (T1D), scleroderma, pemphigus, and lupus.

228. The method of statement 218, wherein said autoimmune disease is type 1 diabetes mellitus (T1D).

229. The method of statement 218, wherein the infectious disease is selected from the group consisting of HIV and a disease caused by a coronavirus infection such as herpes, hepatitis, human papillomavirus, or COVID-19.

230. The method of statement 218, wherein the infectious disease is:

i) HIV; or

ii) How it is COVID-19.

Isolation/Expansion Step

231. The method of any one of statements 201 to 230, further comprising isolating the population of T cells from the subject or from a sample derived from the subject,

Optionally, the isolating step is performed:

i) at least 48 hours after administration of the glucocorticoid;

ii) 48 hours to 13 days after administration of glucocorticoids; or

iii) 6 to 48 hours after administration of the glucocorticoid.

232. The method of statement 231, wherein said sample is selected from the group consisting of blood, plasma, tumor biopsy or surgically removed tumor, bone marrow, liver, and adipose or adipose tissue.

233. The method of statements 231 or 232, further comprising expanding the isolated T cells.

234. The method of any one of statements 231 to 233, further comprising activating the isolated T cell with a T cell activator;

optionally wherein said T cell activator is a T cell activator antibody.

Transfection of isolated T cells

235. The method of any one of statements 231-234, further comprising introducing a nucleic acid encoding the protein into the isolated T cell, and culturing the cell under conditions that promote expression of the protein.

236. The method of statement 235, wherein said protein is selected from the group consisting of one of T-cell receptor (TCR), chimeric antibody receptor (CAR), split, universal and programmable CAR (SUPRA-CAR).

237. The method of statement 236, wherein said CAR and/or TCR comprises an antigen binding domain that binds to an antigen selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, Her2.

238. The method of any one of statements 235-237, further comprising expanding the T cells.

239. The method of any one of statements 235-238, further comprising activating the T cell with a T cell activator.

Administration of isolated T cells

240. A method of treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising a T cell isolated according to any one of statements 231-239, any one of statements 408-413, of a therapeutically effective dose to the subject. A method comprising administering the isolated T cells of, or the cell population of statement 414.

241. The method of statement 240, wherein the subject to which the isolated T cells are administered is the same subject as the subject from which the T cells were isolated.

242. The method of statement 240, wherein the subject to which the isolated T cells are administered is a different subject than the subject from which the T cells were isolated.

243. The method of any one of statements 240 to 242, wherein said T cells are administered to a subject by a method selected from the group consisting of: intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, cerebrospinal fluid ( CSF) intra-injection, direct intra-tumor injection, and gel on or near solid tumors.

medical use

244. A glucocorticoid for use in a method according to any one of statements 201 to 243.

245. Use of a glucocorticoid for the manufacture of a medicament for use in a method according to any one of statements 201 to 243.

246. Use of dexamethasone for inducing a population of T cells, wherein the population of T cells is derived by a method according to any one of statements 201 to 243.

AVM-T cell-derived iPSCs

247. A method of producing induced pluripotent stem cells (iPSCs), the method comprising reprogramming the T cells isolated by the method according to any one of statements 231 to 233 to produce iPSCs.

248. The method of statement 247, wherein said reprogramming comprises introducing into the T cell one or more expression cassettes encoding Oct3/4, Klf4, Sox2, and C-myc.

249. The method of statement 247, wherein said reprogramming comprises introducing mRNA encoding Oct3/4, KLF4, Sox2, and c-myc into the T cell.

250. The method of statements 248 or 249, wherein the reprogramming comprises one or more expression cassettes encoding one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28. The method further comprising the step of introducing into the T cell.

251. The method of statements 248 or 249, wherein said reprogramming comprises introducing one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28 encoding mRNA into a T cell. A method comprising additional steps.

252. The method of any one of statements 247-251, further comprising inducing differentiation of iPSCs.

253. The method of statement 252, wherein said iPSCs differentiate into T cells.

254. A method of producing a population of T cells, said method comprising differentiating iPSCs produced by the method according to any one of statements 247 to 251 into a NKT cell lineage.

AVM-T cells and AVM-dendritic cells

255. The method of any one of statements 201 to 230, wherein the glucocorticoid receptor (GR) modulator also induces a population of NKT cells in the subject,

Optionally, said NKT cell is a method as defined in any one of statements 102 to 108.

256. The method of any one of statements 201-230 or 255, wherein the glucocorticoid receptor (GR) modulator also activates a population of dendritic cells in the subject,

Optionally, said dendritic cell is a method as defined in any one of statements 302-304.

- - -

AVM-dendritic cells

301. A method of producing a population of activated dendritic cells, the method comprising administering to a subject a glucocorticoid receptor (GR) modulator or an ICAM3 modulator at a dose equivalent to approximately at least 6 mg/kg human equivalent dose (HED) of dexamethasone base comprising the steps of

wherein said glucocorticoid receptor (GR) modulator or ICAM3 modulator induces a population of dendritic cells in a subject.

Dendritic cell marker expression

302. The method of statement 301, wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the population of dendritic cells express CD11b.

303. The method of any one of statements 301 to 302, wherein the dendritic cells are CD11b+/very bright,

Optionally, said expression level is determined relative to an average expression level in a population of reference dendritic cells derived from a common source that has not been contacted with a glucocorticoid receptor (GR) modulator.

304. The method of any one of statements 302-303, wherein said expression is measured by flow cytometry, optionally wherein said flow cytometry is performed using the equipment, reagents and/or conditions described herein (taken alone or in combination). How to be.

glucocorticoids

305. The method of any one of statements 301-304, wherein the glucocorticoid receptor (GR) modulator is a glucocorticoid, optionally wherein the glucocorticoid is dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone, budesonide , betamethasone, flumethasone and beclomethasone.

306. The method of statement 305, wherein the glucocorticoid is selected from the group consisting of dexamethasone, betamethasone, and methylprednisone, preferably the glucocorticoid is dexamethasone or betamethasone.

307. The method of any one of statements 305-306, wherein the glucocorticoid is dexamethasone base, dexamethasone sodium phosphate, dexamethasone hemisuccinate, dexamethasone sodium succinate, dexamethasone succinate, dexamethasone isonicotinate, dexamethasone-21-acetate, Dexamethasone phosphate, dexamethasone-21-phosphate, dexamethasone tebutate, dexamethasone-17-valerate, dexamethasone acetate monohydrate, dexamethasone pivalate, dexamethasone palmitate, dexamethasone-21-palmitate, dexamethasone dipropionate, dexamethasone dipropionate , dexamethasone acetate anhydrous, dexamethasone-21-phenylpropionate, dexamethasone-21-sulfobenzoate, dexamethasone hemo-sulfate, dexamethasone sulfate, dexamethasone beloxyl, dexamethasonic acid, dexamethasone acefurate, dexamethasone carboxylate, dexamethasone carboxylate , dexamethasone 21-phosphate disodium salt, dexamethasone mesylate, dexamethasone linoleate, dexamethasone glucoside, dexamethasone glucuronide, dexamethasone iodine acetate, dexamethasone oxetanone, carboxymethylthio-dexamethasone, dexamethasone epoxetoxime, dexamethasone epoxide , dexamethasone linoleidate, dexamethasone methylorthovalerate, dexamethasone spermine, 6-hydroxy dexamethasone, dexamethasone tributyl acetate, dexamethasone aspartic acid, dexamethasone galactopyranose, dexamethasone hydrochloride, hydroxydexamethasone, carboxydexamethasone Desoxydexamethasone, dexamethasone butazone, dexamethasone cyclodextrin, dihydrodexamethasone, oxo-dexamethasone, propionyloxy dexamethasone, dexamethasone galactodi, dexamethasone isonicotinate, dexamethasone sodium hydrogen phosphate tridecyldecyldecyldecyldecide, dexamethasone Late, dexamethasone crotonate, dexamethasone methanesulfonate, dexamethasone butyl acetate, dehydro dexamethasone, dexamethasone isothiocyanatoethyl) thioether, dexamethasone bromoacetate, dexamethasone hemiglutarate, deoxy A method selected from the group consisting of dexamethasone, dexamethasone chlorambusylate, dexamethasone melpalanate, formyloxy dexamethasone, dexamethasone butyrate, dexamethasone laurate, dexamethasone acetate, and any combination treatment containing the form of dexamethasone.

308. The method of statement 307, wherein said dexamethasone is dexamethasone sodium phosphate.

Glucocorticoid Dosage

309. The glucocorticoid of any one of statements 301 to 308, wherein the glucocorticoid is administered at a dose equivalent to about:

i) at least 6-12 mg/kg human equivalent dose (HED) of dexamethasone base;

ii) at least 6 mg/kg human equivalent dose (HED) of dexamethasone base;

iii) at least 12 mg/kg human equivalent dose (HED) of dexamethasone base;

iv) at least 15 mg/kg human equivalent dose (HED) of dexamethasone base;

v) at least 18 mg/kg human equivalent dose (HED) of dexamethasone base;

vi) at least 24 mg/kg human equivalent dose (HED) of dexamethasone base;

vii) 15 mg/kg human equivalent dose (HED) of dexamethasone base;

viii) 24 mg/kg human equivalent dose (HED) of dexamethasone base;

ix) 30 mg/kg human equivalent dose (HED) of dexamethasone base;

x) 45 mg/kg human equivalent dose (HED) of dexamethasone base; or

xi) The human equivalent dose (HED) of dexamethasone base takes a mg/kg value in a range of mg/kg values, wherein the range is limited by two of the mg/kg values given in parts i) to x) above.

310. The method of any one of statements 301-309, wherein said glucocorticoid is administered in a single acute dose, or in a total dose given over about 72 hours.

311. The method of any one of statements 301-310, wherein the method comprises administering one or more additional doses of the glucocorticoid.

312. The method of statement 311, wherein the one or more additional doses are administered as follows:

i) 24 to 120 hours after prior glucocorticoid administration;

ii) 24 to 48 hours after prior glucocorticoid administration;

iii) 72 to 120 hours after prior glucocorticoid administration;

iv) every 24, 48, 72, 96, 120, 144, or 168 hours after the first glucocorticoid administration;

v) once every 2 weeks after the first glucocorticoid administration;

vi) once a month after the first glucocorticoid administration; or

vii) Twice weekly after the first glucocorticoid administration.

Dendritic cell activation

313. The method of any one of statements 301-312, further comprising administering to the subject a dendritic cell activator.

314. The method of statement 313, wherein the dendritic cell activator is administered within 48 hours or before or after 48 hours of administration of the glucocorticoid.

object

315. The method according to any one of statements 301 to 314, wherein the subject is a mammal, preferably the subject is a human.

316. The method of any of statements 301-315, wherein the subject has, is suspected of having, or has been diagnosed with a disease selected from the group consisting of cancer, an autoimmune disease or an infectious disease.

317. The method of statement 316, wherein said cancer is a solid tumor cancer.

318. The method of statement 316, wherein said cancer is selected from the group consisting of: squamous cell carcinoma (eg epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung; peritoneal cancer; hepatocellular carcinoma; stomach or abdominal cancer, including gastrointestinal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; liver tumor; breast cancer; colorectal cancer; rectal cancer; colorectal cancer; endometrial or uterine cancer; salivary adenocarcinoma; kidney or kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; hepatocarcinoma; anal carcinoma; penile carcinoma; and head and neck cancer.

319. The method of statement 318, wherein the cancer is a lymphoma, preferably a B cell lymphoma, a T cell lymphoma, or a non-Hodgkin's lymphoma.

320. The method of any one of statements 317 to 319, wherein said dendritic cells treat cancer through tumor infiltration.

321. The method of statement 320, wherein said dendritic cells treat cancer through the release of immune activating cytokines.

322. The method according to any one of statements 320 to 321, wherein said dendritic cells promote infiltration of other immune cells, such as T cells, into the tumor.

323. The method according to any one of statements 320 to 322, wherein said dendritic cells promote tumor necrosis.

324. The method of statement 316, wherein said autoimmune disease is selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes mellitus (T1D), scleroderma, pemphigus, and lupus.

325. The method of statement 316, wherein said autoimmune disease is type 1 diabetes mellitus (T1D).

326. The method of statement 316, wherein the infectious disease is selected from the group consisting of HIV and a disease caused by a coronavirus infection such as herpes, hepatitis, human papillomavirus, or COVID-19.

327. The method of statement 316, wherein the infectious disease is:

i) HIV; or

ii) How it is COVID-19.

Isolation/Expansion Step

328. The method of any one of statements 301 to 327, further comprising isolating the population of dendritic cells from the subject or from a sample derived from the subject,

Optionally, the isolating step is performed by:

iv) at least 48 hours after administration of the glucocorticoid;

v) 48 hours to 13 days after administration of glucocorticoids; or

vi) 6 to 48 hours after administration of the glucocorticoid.

329. The method of statement 328, wherein said sample is selected from the group consisting of blood, plasma, tumor biopsy or surgically removed tumor, bone marrow, liver, and adipose or adipose tissue.

330. The method of statements 328 or 329, further comprising expanding the isolated dendritic cells.

331. The method of any one of statements 328-330, further comprising expanding the isolated dendritic cells with a dendritic cell activator.

Transfection of isolated dendritic cells

332. The method of any one of statements 328 to 331, further comprising introducing a nucleic acid encoding the protein into the isolated dendritic cell, and culturing the cell under conditions that promote expression of the protein.

333. The method of statement 332, wherein said protein is selected from the group consisting of one or more of T-cell receptor (TCR), chimeric antibody receptor (CAR), split, universal and programmable CAR (SUPRA-CAR).

334. The method of statement 333, wherein said CAR and/or TCR comprises an antigen binding domain that binds to an antigen selected from the group consisting of: CD19, CD20, CD22, GD2, CD133, EGFR, GPC3, CEA, MUC1, mesothelin, IL-13R, PSMA, ROR1, CAIX, Her2.

335. The method of any one of statements 332 to 334, further comprising expanding the dendritic cells.

336. The method of any one of statements 332 to 335, further comprising activating the dendritic cells with a dendritic cell activator.

Administration of isolated dendritic cells

337. A method of treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising: a dendritic cell isolated according to any one of statements 328-336 of a therapeutically effective dose to the subject, any one of statements 415-420 A method comprising administering the isolated dendritic cells of, or the cell population of statement 421.

338. The method of statement 337, wherein the subject to which the isolated dendritic cells are administered is the same subject from which the dendritic cells were isolated.

339. The method of statement 337, wherein the subject to which the isolated dendritic cells are administered is a different subject than the subject from which the dendritic cells were isolated.

340. The method of any one of statements 337-339, wherein the dendritic cells are administered to the subject by a method selected from the group consisting of: intravenous injection, intraperitoneal injection, intralymphatic injection, intrathecal injection, cerebrospinal fluid ( CSF) intra-injection, direct intra-tumor injection, and gel on or near solid tumors.

medical use

341. A glucocorticoid for use in a method according to any one of statements 301 to 340.

342. Use of a glucocorticoid for the manufacture of a medicament for use in a method according to any one of statements 301 to 340.

343. Use of dexamethasone for inducing a population of dendritic cells, wherein the population of dendritic cells is derived by a method according to any one of statements 301 to 340.

AVM-dendritic cell-derived iPSCs

344. A method of producing induced pluripotent stem cells (iPSCs), the method comprising reprogramming the dendritic cells isolated by the method according to any one of statements 328 to 330 to produce iPSCs.

345. The method of statement 344, wherein said reprogramming comprises introducing into the dendritic cell one or more expression cassettes encoding Oct3/4, Klf4, Sox2, and C-myc.

346. The method of statement 344, wherein said reprogramming comprises introducing Oct3/4, KLF4, Sox2, and c-myc encoding mRNA into the dendritic cell.

347. The method of statements 345 or 346, wherein the reprogramming comprises one or more expression cassettes encoding one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28. A method further comprising introducing into a dendritic cell.

348. The method of statements 345 or 346, wherein said reprogramming comprises introducing one or more of Sox1, Sox3, Sox15, Klf1, Klf2, Klf5, L-myc, N-myc, Nanog, and/or LIN28 encoding mRNA into a dendritic cell. How to include more.

349. The method of any one of statements 344 to 348, further comprising inducing differentiation of the iPSCs.

350. The method of statement 349, wherein said iPSCs differentiate into dendritic cells.

351. A method of producing a population of dendritic cells, said method comprising differentiating iPSCs produced by the method according to any one of statements 344 to 348 into a dendritic cell lineage.

AVM-T cells and AVM-dendritic cells

352. The method of any one of statements 301 to 327, wherein the glucocorticoid receptor (GR) modulator also induces a population of NKT cells in the subject,

Optionally, said NKT cells are as defined by any one of statements 102 to 108.

353. The method of any one of statements 301 to 327 or 352, wherein the glucocorticoid receptor (GR) modulator also activates a population of T cells in the subject,

Optionally, said T cell is a method as defined by any one of statements 202-205.

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401. An isolated natural killer T cell (NKT cell) or population of natural killer T cells (NKT cell) produced by the method according to any one of statements 101 to 159.

402. An isolated natural killer T cell (NKT cell), wherein the cell expresses CD3, and

i) express CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;

ii) an isolated NKT cell, characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta.

403. The NKT cell or precursor thereof of statement 402, wherein said NKT cell or precursor thereof was isolated from the subject, and wherein said NKT cell or NKT cell was isolated in vivo prior to isolation. or in vitro contacted with a high dose glucocorticoid receptor (GR) modulator after isolation, wherein the CD3 expression level is at least 2-fold higher than the mean level of CD3 expression in a reference NKT cell population from a subject not contacted with the GR modulator NKT cells.

404. The isolated NKT cell of statement 403, wherein the CD3 expression level of said isolated NKT cell and said reference NKT cell population is determined by flow cytometry.

405. The isolated NKT cell of statement 404, wherein said flow cytometry is performed using the equipment, reagents and/or conditions (taken in isolation or in combination) described herein.

406. The method of any one of statements 403-405, wherein the CD3 expression level of the isolated NKT cells is at least 3 greater than the average level of CD3 expression in a population of reference NKT cells from a subject not contacted with a glucocorticoid receptor (GR) modulator. fold, at least 4 fold, or at least 5 fold higher isolated NKT cells.

407. An isolated population of natural killer T cells (NKT cells), wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells comprise:

i) express CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;

ii) an isolated population of NKT cells characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta.

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408. An isolated T cell or population of T cells produced by a method according to any one of statements 201 to 254.

409. An isolated T cell, wherein said cell expresses CD3,

i) express CD4, CD45, and/or CD49b and/or

ii) does not express CD8;

optionally wherein said cell expresses TCR gamma/delta.

410. The method of statement 409, wherein said T cells or precursors thereof have been isolated from a subject, and wherein said T cells or precursors thereof are in vivo. pre-isolation or in vitro An isolated T cell contacted with an isolated high dose glucocorticoid receptor (GR) modulator, wherein the CD3 expression level is at least 2-fold higher than the average level of CD3 expression in a reference T cell population from a subject not contacted with the GR modulator.

411. The isolated T cell of statement 410, wherein the CD3 expression level of said isolated T cell and said population of reference T cells is determined by flow cytometry.

412. The isolated T cell of statement 411, wherein said flow cytometry is performed using the equipment, reagents and/or conditions (taken in isolation or in combination) described herein.

413. The CD3 expression level of any of statements 410-412, wherein the CD3 expression level of the isolated T cells is at least less than the average level of CD3 expression in the population of reference T cells from a subject not contacted with a glucocorticoid receptor (GR) modulator. 3 fold, at least 4 fold, or at least 5 fold higher isolated T cells.

414. A population of isolated T cells, wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells are

i) express CD3, CD4, CD45, and/or CD49b;

ii) does not express CD8;

wherein the CD3 expression level is at least 3-fold, at least 4-fold, or at least 5-fold higher than the average level of CD3 expression in a reference T cell population from a subject not contacted with a glucocorticoid receptor (GR) modulator;

optionally wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express TCR gamma/delta.

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415. An isolated dendritic cell or population of dendritic cells produced by a method according to any one of statements 301 to 351.

416. An isolated dendritic cell, wherein said cell expresses CD11b.

417. The dendritic cell or precursor thereof of statement 416 is isolated from the subject, wherein the dendritic cell or precursor of the dendritic cell is in vivo. pre-isolation or in vitro Isolated dendritic cells contacted with a high dose glucocorticoid receptor (GR) modulator after isolation, wherein said CD11b expression level is at least 2-fold higher than the average level of CD11b expression in a population of reference dendritic cells from a subject not contacted with a GR modulator .

418. The isolated dendritic cell of statement 417, wherein the CD11b expression level of the population of said isolated dendritic cells and said reference dendritic cells is determined by flow cytometry.

419. The isolated dendritic cell of statement 418, wherein said flow cytometry is performed using the equipment, reagents and/or conditions (taken in isolation or in combination) described herein.

420. The CD11b expression level of any of statements 417-419, wherein the CD11b expression level of the isolated dendritic cells is at least 3 greater than the average level of CD11b expression in a population of reference dendritic cells from a subject not contacted with a glucocorticoid receptor (GR) modulator. an isolated dendritic cell that is fold, at least 4 fold, or at least 5 fold higher.

421. A population of isolated dendritic cells, wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD11b; wherein the CD11b expression is at least 3-fold, at least 4-fold, or at least 5-fold higher than the average level of CD11b expression in a reference dendritic population from a subject not contacted with a cellular glucocorticoid receptor (GR) modulator An isolated population of dendritic cells.

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422. A glucocorticoid for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, comprising administering the glucocorticoid to the subject at about 6-45 mg/kg human equivalent dose (HED) of dexamethasone base; including administration in equivalent doses,

wherein said glucocorticoid has at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of cells:

i) express CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;

ii) a glucocorticoid inducing a population of NKT cells, characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta.

423. A glucocorticoid for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, comprising administering the glucocorticoid to the subject at about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone base; including administration in equivalent doses,

wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells express CD3, wherein the CD3 expression level is from a subject not contacted with a glucocorticoid receptor (GR) modulator. A glucocorticoid that induces a population of T cells, characterized in that it is at least 3-fold, at least 4-fold, or at least 5-fold higher than the average level of CD3 expression in the population of reference T cells.

424. A glucocorticoid for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, wherein the method is equivalent to about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone base to a glucocorticoid subject. comprising administering in a dose,

at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells of the glucocorticoid express CD11b; wherein the CD11b expression level is at least 3-fold, at least 4-fold, or at least 5-fold higher than the average level of CD11b expression in a reference dendritic cell population from a subject not contacted with a glucocorticoid receptor (GR) modulator. glucocorticoids that activate the population of

425. A glucocorticoid for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, comprising administering the glucocorticoid to the subject at about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone base; including administration in equivalent doses,

The glucocorticoid is:

i) inducing a population of natural killer T cells (NKT cells) as defined in any one of statements 101 to 159;

ii) inducing a population of T cells as defined in any one of statements 201 to 254;

iii) a glucocorticoid that activates a population of dendritic cells as defined in any one of statements 301 to 351.

426. A method of treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising:

i) an NKT cell isolated according to any one of statements 136 to 144, an isolated NKT cell according to any one of statements 401 to 406, or a cell population according to statement 407;

ii) a T cell isolated according to any one of statements 231-239, an isolated T cell according to any one of statements 408-413, or a cell population of any one of statements 414; and/or

iii) a method comprising administering to the subject an isolated dendritic cell according to any one of statements 328 to 336, an isolated dendritic cell according to any one of statements 415 to 420, or a cell population according to statement 421.

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501. A method of treating a disease due to a coronavirus infection in a subject, the method comprising administering to the subject a glucocorticoid receptor (GR) modulator at a dose equivalent to approximately at least 6 mg/kg human equivalent dose (HED) of dexamethasone base. how to include it.

502. The method of statement 501, wherein the glucocorticoid receptor (GR) modulator is a glucocorticoid.

503. The method of statement 501, wherein the glucocorticoid receptor (GR) modulator is dexamethasone or betamethasone.

504. The method of any of statements 501-503, wherein the glucocorticoid receptor modulator is administered at a dose equivalent to approximately at least 18 mg/kg human equivalent dose (HED) of dexamethasone base.

505. The method of any one of statements 501 to 504, wherein the glucocorticoid receptor modulator is administered at a dose equivalent to about 18 mg/kg to 30 mg/kg human equivalent dose (HED) of dexamethasone base.

506. The method of any of statements 501 to 505, wherein the disease is COVID-19.

507. The method of any one of statements 501 to 506, wherein the glucocorticoid receptor modulator is:

i) inducing a population of natural killer T cells (NKT cells) as defined in any one of statements 101 to 159;

ii) inducing a population of T cells as defined in any one of statements 201 to 254;

iii) a method of activating a population of dendritic cells as defined in any one of statements 301 to 351.

508. The method of statement 507, wherein the NKT cells engulf and kill the coronavirus and/or activate other innate and adaptive immune cells to treat a disease.

509. A glucocorticoid receptor (GR) modulator for use in a method according to any one of statements 501 to 508.

510. Use of a glucocorticoid receptor (GR) modulator for the manufacture of a medicament for use in a method according to any one of statements 501 to 508.

Claims (40)

A method of producing a population of natural killer T cells (NKT cells), the method comprising administering to a subject a glucocorticoid receptor (GR) modulator at a dose equivalent to approximately at least 6 mg/kg human equivalent dose (HED) of dexamethasone base A method comprising steps. The method of claim 1 , wherein the population of NKT cells comprises at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells:
i) express CD3; and
ii) express CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;
iii) does not express C-kit, B220, FoxP3, and/or TCR alpha/beta. 3. The method of claim 2, wherein said NKT cells express:
i) CD3, CD4, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and TCR gamma/delta; or
ii) CD3, CD45, and CD56. 4. The method of claim 2 or 3, wherein the NKT cell comprises:
i) does not express C-kit, B220, FoxP3, or TCR alpha/beta;
ii) does not express CD8,
iii) express CD4 and CD8;
iv) express Ly6G and TCR gamma/delta;
v) CD3+very bright and/or CD45+/dark and/or CD56+. 5. The glucocorticoid receptor (GR) modulator according to any one of claims 1 to 4, wherein the glucocorticoid receptor (GR) modulator is a glucocorticoid, optionally the glucocorticoid is dexamethasone, hydrocortisone, methylprednisolone, prednisone, prednisolone, prednylidene, cortisone. , budesonide, betamethasone, flumethasone and beclomethasone. 6. The method of claim 5, wherein the glucocorticoid is selected from the group consisting of dexamethasone, betamethasone, and methylprednisone. 7. The method of claim 6, wherein the glucocorticoid is dexamethasone or betamethasone. 8. The method according to any one of claims 5 to 7, wherein said dexamethasone is dexamethasone sodium phosphate. 9. The method of any one of claims 1-8, wherein said glucocorticoid is administered at a dose equivalent to about:
i) at least 6-12 mg/kg human equivalent dose (HED) of dexamethasone base;
ii) at least 6 mg/kg human equivalent dose (HED) of dexamethasone base;
iii) at least 12 mg/kg human equivalent dose (HED) of dexamethasone base;
iv) at least 15 mg/kg human equivalent dose (HED) of dexamethasone base;
v) at least 21 mg/kg human equivalent dose (HED) of dexamethasone base;
vi) at least 24 mg/kg human equivalent dose (HED) of dexamethasone base;
vii) 15 mg/kg human equivalent dose (HED) of dexamethasone base;
viii) 24 mg/kg human equivalent dose (HED) of dexamethasone base; or
ix) 45 mg/kg human equivalent dose (HED) of dexamethasone base. 10. The method of any one of claims 1-9, wherein said glucocorticoid is administered in a single acute dose, or in a total dose given over about 72 hours. 11. The method of any one of claims 1-10, wherein the method comprises administering one or more additional doses of the glucocorticoid. 12. The method of any one of claims 1-11, further comprising administering to the subject a NKT cell activator. The method of claim 12, wherein the NKT cell activator is administered within 48 hours or before or after 48 hours after administration of the glucocorticoid. 14. The method of any one of claims 1-13, wherein the subject has, is suspected of having, or is diagnosed with a disease selected from the group consisting of cancer, an autoimmune disease, or an infectious disease. 15. The method of claim 14, wherein said cancer is a solid tumor cancer. 16. The method according to claim 15, wherein said cancer is a lymphoma, preferably a B-cell lymphoma, a T-cell lymphoma, or a non-Hodgkin's lymphoma. 17. The method according to any one of claims 14 to 16, wherein said NKT cells are infiltrated through tumor infiltration. How to treat cancer. 18. The method according to any one of claims 14 to 17, wherein said NKT cells promote infiltration of other immune cells into the tumor. 19. The method according to any one of claims 14 to 18, wherein said NKT cells directly kill cancer cells through CD1d-induced apoptosis. 20. The method of any one of claims 14-19, wherein said NKT cells cause tumor necrosis, thereby treating cancer. 15. The method of claim 14, wherein said autoimmune disease is selected from the group consisting of multiple sclerosis, systemic sclerosis, amyotrophic lateral sclerosis, type 1 diabetes mellitus (T1D), scleroderma, pemphigus, and lupus. 15. The method of claim 14, wherein the infectious disease is a disease caused by a coronavirus infection such as HIV or COVID-19. 23. The method of any one of claims 1-22, further comprising isolating the population of NKT cells from the subject or from a sample derived from the subject,
Optionally, the isolating step
i) at least 48 hours after administration of the glucocorticoid; or
ii) from 48 hours to 13 days after administration of the glucocorticoid. 24. The method of claim 23, wherein said sample is selected from the group consisting of blood, plasma, tumor biopsy or surgically removed tumor, bone marrow, liver, and adipose or adipose tissue. 25. The method of claim 23 or 24, further comprising expanding the isolated NKT cells. 26. The method of any one of claims 23-25, further comprising activating the isolated NKT cells with an NKT cell activator.
Optionally, said NKT cell activator is selected from cytokines, chemokines, growth factors, and/or NKT modulators. 26. The method of any one of claims 23-25, further comprising introducing a nucleic acid encoding a protein into the isolated NKT cell, and culturing the cell under conditions that promote expression of the protein. . 28. The method of claim 27, wherein said protein is selected from the group consisting of T-cell receptor (TCR), chimeric antibody receptor (CAR), and one or more of split, universal and programmable CAR (SUPRA-CAR). 29. The method of any one of claims 23-28, further comprising expanding the NKT cells. 30. The method of any one of claims 23-29, further comprising activating the NKT cells with an NKT cell activator. 31. A method of treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising administering to the subject a therapeutically effective dose of the isolated NKT cells of any one of claims 1-30. 32. A glucocorticoid for use in the method according to any one of claims 1 to 31. 32. Use of a glucocorticoid for the manufacture of a medicament for use in a method according to any one of claims 1 to 31. 34. An isolated natural killer T cell (NKT cell) or population of natural killer T cells (NKT cell) produced by the method according to any one of claims 1-33. An isolated natural killer T cell (NKT cell), said cell expressing CD3,
i) express CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;
ii) does not express C-kit, B220, FoxP3, and/or TCR alpha/beta;
Optionally, the isolated NKT cells are CD3+very bright and/or CD45+/dark and/or CD56+. 36. The method of claim 35, wherein the NKT cells or precursors thereof have been isolated from a subject, and the NKT cells or precursors of NKT cells are in vivo prior to isolation. or isolated NKT contacted with a high-dose glucocorticoid receptor modulator in vitro after isolation, wherein said CD3 expression level is at least 2-fold higher than the average level of CD3 expression in a reference NKT cell population from a subject not contacted with a glucocorticoid receptor modulator cell. 37. The isolated NKT cell of claim 36, wherein the CD3 expression level of said isolated NKT cell and said reference NKT cell population is measured by flow cytometry. 38. The method of claim 36 or 37, wherein the CD3 expression level of the isolated NKT cells is at least 3-fold, at least 4-fold greater than the average level of CD3 expression in a reference NKT cell population from a subject not contacted with a glucocorticoid receptor modulator. , or at least 5-fold higher isolated NKT cells. An isolated population of natural killer T cells (NKT cells), wherein at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of the cells comprise:
i) express CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;
ii) does not express C-kit, B220, FoxP3, and/or TCR alpha/beta;
optionally said at least 60, 70, 80, 90, 95, 96, 97, 98, or 99 % of the cells are CD3 + very bright and/or CD45 + / dark and/or CD56 + group. A glucocorticoid for use in a method of treating cancer, an autoimmune disease, or an infectious disease in a subject, the method comprising administering the glucocorticoid to the subject at a dose equivalent to about 6 - 45 mg/kg human equivalent dose (HED) of dexamethasone base including administering with
wherein said glucocorticoid has at least 60, 70, 80, 90, 95, 96, 97, 98, or 99% of cells:
i) express CD3, CD4, CD8, CD45, CD49b, CD56, CD62L, NK1.1, Ly6G, Sca1, and/or TCR gamma/delta;
ii) a glucocorticoid that induces a population of NKT cells characterized in that it does not express C-kit, B220, FoxP3, and/or TCR alpha/beta.

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